Designing Triple Helical Fragments: The Crystal Structure of the

DOI: 10.1021/cg1009048

Designing Triple Helical Fragments: The Crystal Structure of the Undecamer d(TGGCCTTAAGG) Mimicking T 3 AT Base Triplets†

2010, Vol. 10 4622–4629

Kristof Van Hecke,*,‡ Koen Uytterhoeven,‡ Arnout Voet,# Marc De Maeyer,# and Luc Van Meervelt‡ ‡

Biomolecular Architecture and BioMacS, Katholieke Universiteit Leuven, Department of Chemistry, Celestijnenlaan 200F, B-3001 Leuven, Belgium, and #Biomolecular Modeling and BioMacS, Katholieke Universiteit Leuven, Department of Chemistry, Celestijnenlaan 200G, B-3001 Leuven, Belgium Received July 7, 2010; Revised Manuscript Received August 25, 2010

ABSTRACT: Crystal engineering techniques that mimic triple helical fragments in the crystal lattice are able to reveal interesting features of short triple Pu 3 PuPy helical fragments. The nonamer d(GCGAATTCG) and decamer d(GGCCAATTGG), containing respectively one and two overhanging guanines, were designed to form G 3 GC triplets in the crystal packing. To introduce a third subsequent T 3 AT triplet and thus a Pu 3 PuPy to Py 3 PuPy triplet transition, the decamer was further extended with one overhanging thymine residue. The resulting undecamer d(TGGCCTTAAGG) forms an extra parallel Hoogsteen T 3 AT triplet at one end of the duplex and an unexpected (swung out over 270°), antiparallel reverse-Hoogsteen T 3 AT triplet at the other end of the duplex, providing detailed X-ray structural models of parallel and antiparallel T 3 AT triplets. The derived parameters allow for the construction of larger parallel and antiparallel triple helical models.

1. Introduction The association of a nucleic acid double helix and a singlestranded oligonucleotide results in so-called triple helices, which can be characterized by their base complementarity and the relative orientations of their sugar-phosphate backbone.1-3 Hydrogen bonds are formed between the available functional groups of one of the base pairs in a Watson-Crick double helix and a third (Hoogsteen) strand located in the major groove of the double helix. This specific sequence recognition allows the targeting of double-stranded DNA sequences to exclude DNA binding proteins4-6 and to direct single-site cleavage in chromosomal DNA.7 Considering genetic recombination in vivo, triplex formation has also been presented as a mechanism for the alignment of homologue sequences.8,9 Furthermore, triple-helix formation has been observed at different positions on nucleosomal DNA10 and found to inhibit DNA gyrase activity,11 to mediate sitespecific genome modification,12 and even to repair genes.13 When an oligonucleotide hybridizes with a double-stranded DNA (dsDNA) to form a stable triplex, this feature can be exploited in the antigene strategy: specific DNA regions can be targeted in order to hamper the mRNA synthesis, which might be a more efficient approach than targeting proteins, which are the result of the later translation step in gene expression. Such exogenous triplex forming oligonucleotides (TFOs) have already been developed, but the physiological stability of these triplexes forms the main challenge in designing novel TFOs.14 Despite the importance of triple helices, only limited X-ray structural information is available. Some conformational information about these species is available from spectroscopic studies in solution and from fiber diffraction: NMR structures exist for Pu 3 PuPy and Py 3 PuPy tracts, containing G 3 GC, T 3 AT,15 Cþ 3 GC,16 and T 3 CG triplets.17 Furthermore, *To whom correspondence should be addressed. Tel: þ3216327477. Fax: þ3216327990. E-mail: [email protected]. † The atomic coordinates and structure factors have been deposited in the Protein Data Bank41 and Nucleic Acid Database42 (PDB and NDB entry codes 3L1Q and NA0392, respectively). pubs.acs.org/crystal

Published on Web 09/13/2010

a triplex between a dsDNA and a locked nucleic acid (LNA), containing a 20 -O,40 -C methylene bridge, has been observed.18 The X-ray structure of a 2:1 peptide nucleic acid-DNA triplex has been reported,19 but crystals formed by nucleic acid triplexes are invariably disordered, at best giving rise to fiber-like diffraction.20 The only crystallographic example of a short parallel DNA triplex is the structure reported by Rhee et al., containing three consecutive Cþ 3 GC, BrU 3 ABrU, CþG 3 C triplets.21 In view of this lack of X-ray structural information on triplex DNA, especially on antiparallel triplexes, we introduced a novel way of exploiting the ability of overhanging bases to form triple helical fragments in the crystal packing.22 This crystal engineering technique has already resulted in detailed models of a parallel Hoogsteen G 3 GC triplet in the crystal lattice of the nonamer d(GCGAATTCG)22,24 and of a parallel Hoogsteen and antiparallel reverse-Hoogsteen (G 3 GC)2 triple helical fragment in the lattice of the decamer d(GGCCAATTGG).23,25 An apparent feature of triple helical fragments, mimicked in the crystal lattice of d(GGCCAATTGG), is the ability to enhance the resolution of the obtained diffraction data. Hence, we have previously reported the d(GGCCAATTGG) decamer in complex with minor groove binders 40 ,6-diamidino-2-phenylindole (DAPI), distamycin, and netropsin to the highest resolution of 1.9, 1.85, and 1.75 A˚, respectively.26-28 The reported structure was constructed by further extending the decamer with one additional overhanging thymine residue. Interaction of these thymines with AT base pairs results in the formation of T 3 AT triplets and hence an extension of the triple helical fragment to three triplets and at the same time the introduction of a Pu 3 PuPy to Py 3 PuPy triplet transition. The central AATT sequence had to be inverted to TTAA in comparison with the nona- and decamer, due to geometric restrictions of the T 3 AT triplet (Figure 1). Crystallization and data collection have previously been described.29 The resulting undecamer d(TGGCCTTAAGG) forms short triple helical fragments in the crystal packing: at one r 2010 American Chemical Society

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Crystal Growth & Design, Vol. 10, No. 10, 2010

Figure 1. Schematic representation of the triple helix formation of the undecamer d(TGGCCTTAAGG). Guanine, adenine, thymine, and cytosine bases are shown in green, red, blue, and yellow, respectively. The sugar-phosphate backbone of the asymmetric unit duplex is shown as a dark gray ribbon. Sugar-phosphate backbones of symmetry equivalent duplexes are shown as light gray, green, and blue ribbons, respectively. Watson-Crick hydrogen bonds are shown in black and (reverse-) Hoogsteen bonds in red.

end of the duplex, the overhanging bases interact in a continuous manner in the major groove of a neighboring helix in the same column, forming a (G 3 GC)2/T 3 AT parallel Hoogsteen triple helix stretch. At the other end of the duplex, the two overhanging guanine bases swing out 180° to form G 3 GC antiparallel reverse-Hoogsteen triplets with another column and the end-standing thymine base swings out 270° to form a T 3 AT antiparallel reverse-Hoogsteen triplet with even another column. Hence, the antiparallel triple helical fragment contains bases from three different asymmetric units. 2. Materials and Methods 2.1. Crystallization. The DNA undecamer d(TGGCCTTAAGG) was purchased from Oswel DNA service (University of Southampton, UK). Two different crystal forms (bar-shaped and hexagonal-shaped blocks), suitable for X-ray diffraction, were obtained by the

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sitting-drop vapor-diffusion method at 16 °C, as previously described.29 2.2. Data Collection and Processing. For both crystal morphologies, data were collected at the EMBL of the DESY synchrotron in Hamburg on a MAR345 imaging plate detector and a liquidnitrogen cryostream at 100 K was used to diminish radiation damage to the crystals. Data were processed with iMOSFLM v0.4.530 and scaled using Scala v3.2.5.31 The latter was used as part of the CCP4.32 An 84.8% complete data set to 2.30 A˚ resolution was obtained for a blockshaped crystal, whereas the hexagonal-shaped crystals diffracted only to 2.50 A˚, but resulting in a 98.5% complete data set. Both data sets were initially merged as both crystals have the same unit cell and space group.29 The resulting Rmerge value of the averaged data set was however higher than those of the individual data sets, which may be caused by the different crystallization conditions, crystal dimensions (absorption effects) and data collection conditions. Therefore, only the complete data set to 2.50 A˚ was used for the structure refinement. Final data collection statistics have been previously published.29 2.3. Structure Solution and Refinement. Both the space group and unit cell parameters of the undecamer d(TGGCCTTAAGG) indicated isomorphism with the decamer d(GGCCAATTGG)2,23 which could be used as a starting model for molecular replacement, despite the inverted central AATT sequence and the missing endstanding thymine residues. A molecular replacement solution was found with the program Phaser v1.3.3.33 The structure was refined with the program Refmac v5.4.006734 and the convergence was cross-validated using a 4.5% Rfree-value test set, giving an initial R-value and Rfree-value of 36.7% and 49.7%, respectively. After the central AATT sequence was mutated to TTAA with the program Coot v0.5.2.,35 the R-value (Rfree) decreased to 26.9% (37.5%). At this stage, the Fo - Fc map showed the density of the overhanging thymine base T20 beneath the G21 residue at the continuous end of the helix (see Figure 1 for residue numbering). Although this position of the T20 base could be clearly distinguished from the difference map, residual density was observed in the immediate environment of the base, pointing to a slight disorder of this endstanding base. Therefore, the T20 base was refined with an occupancy of value 0.5. At the other end of the helix, the overhanging bases G1 and G2 show a similar interaction as in the decamer; that is, they are twisted away and interact with a neighboring duplex. However, as no space is left for a third overhanging base to interact with the same adjacent duplex, a similar interaction for thymine T0 is not possible. A detailed analysis of the Fo - Fc map in this region showed density pointing toward an AT base pair of another neighboring helix (Figure 2). Positional refinement with the extra overhanging bases T0 and T20 decreased the R-factor to 24.13% (33.8%). None of the hydrogen bonds of the Watson-Crick base pairs of the central octamer, nor the (reverse-)Hoogsteen hydrogen bonds of the overhanging bases were restrained. A total of 44 water molecules were carefully added with the program Coot v0.5.2.35 Neither mono- or bivalent ions nor spermine molecules could be univocally identified. The final R- and Rfree-values were 20.6% and 28.3%, respectively. Noticing the limited amount of reflections present, especially for calculating the Rfree-value, the last refinement cycles were repeated, including all reflections and omitting the calculation of the Rfree-value, leading to a final R-value of 22.3%. Further refinement details are listed in Table 1. The first strand residues are labeled T0 to G10 in the 50 -30 direction, and the second strand residues are labeled T20 to G30 in the 50 -30 direction. Helical parameters in accordance with the Tsukuba Workshop guidelines36 and torsion angles were calculated with the program 3DNA.37 For the triple helical structures, rise and twist are calculated based on the Watson-Crick duplexes, unless stated otherwise. All molecular figures were created using the PyMOL software (DeLano, W.L. The PyMOL Molecular Graphics System (2002) on World Wide Web http://www.pymol.org). 2.4. Modeling. Models of both parallel and antiparallel triple helices were constructed, based on the final X-ray structure, and the sugar-phosphate backbone was further optimized. The reported structure provides only partially the necessary twist and rise helical parameters for construction of both models; that is, the twist and

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Figure 2. Left: Stick representation after initial refinement with the model decamer d(GGCCTTAAGG), showing the Fo - Fc difference Fourier map density (1.5σ contour level) for the missing overhanging thymine bases T0 and T20 of the undecamer d(TGGCCTTAAGG). Right: Stick representation of the final undecamer d(TGGCCTTAAGG). Table 1. Refinement Statistics for the Undecamer d(TGGCCTTAAGG) space group asymmetric unit resolution range (A˚) number of independent reflections number of atoms nucleic acid waters (treated as O) final R-value (all data) (%) rms deviation from restraint target value bond lengths (A˚) angles (°) distances from restraint planes (A˚) mean B-values (A˚2) DNA atoms solvent atoms

P212121 d(TGGCCTTAAGG)2 32.36-2.50 2192

The antiparallel triple helix was generated in an analogous manner. The 12-base triplet helices were further optimized as described for the refinement of the six base triplets.

3. Results and Discussion 450 44 22.3 0.016 2.8 0.011 50.3 48.9

rise of the G 3 GC/T 3 AT step is not known from the reported structure. Hence, for the construction of complete models of 12 base triplets, one “trimer”, consisting of the parallel G22 3 G30C3, G21 3 G29C4, and T20 3 A28T5 triplets was docked onto the same “trimer”. The following procedure was developed using the Brugel program.38 First, the two trimers were aligned onto the Z-axis. The lower trimer was subsequently docked as a rigid body by allowing rotations and translations in all x-, y-, and z-directions. The step size was 1° and 0.1 A˚ for rotation and for translation, respectively. At each step, the nonbonded energy between the two docking parts was calculated and a rank-ordered energy sorted list was created. The best scoring structures were retained for 500 steps steepest descend followed by 500 steps of conjugated gradient energy minimization. A constraint was applied on the positions of the bases, allowing the sugars to adapt to the docked positions. This resulted in a triple helix, consisting of six base triplets. Subsequently, a 12-base triplet parallel helix was constructed by aligning the lower three triplets of the obtained six base triplet helix to the upper three triplets through least-squares fitting (of the bases) onto a second helix. This procedure was repeated until the desired length of the sequence [(G 3 GC)2 /T 3 AT]4 was obtained.

3.1. Overall Structure. Because of the mediocre resolution of the reported structure, care should be taken not to overinterpret the results. Nevertheless, some essential features can be distinguished. In Figure 2, an overall representation of the undecamer d(TGGCCTTAAGG) is given. In comparison with the nonamer d(GCGAATTCG)22 and the decamer d(GGCCAATTGG),23 the central octamer d(CCTTAAGG) of the undecamer has an inverted central TTAA sequence and is flanked at both ends by three overhanging bases, two guanines and one thymine, which interact with symmetry equivalent duplexes to form triple helical fragments. Because of the TGG overhanging sequence not only the GC base pairs but also two of the four AT base pairs are influenced by triple helix formation. As noticed for the related decamer d(GGCCAATTGG),23 the sugar-phosphate backbone of the two overhanging guanines G21, G22 and thymine T20 in addition, at one end of the central octamer of d(TGGCCTTAAGG) (Figures 1 and 2), interacts in a continuous manner in the major groove of a neighboring helix in the same column. At the other end G1 and G2 are rotated over approximately 180° to interact with another column. The central octamer forms a B-DNA double helix, consisting of normal Watson-Crick base pairs. The average helical twist for the octamer base pairs is 35.4° or 10.2 residues per turn with an average helical rise of 3.3 A˚. Analogous to the base pairs of the central octamer, all overhanging guanines are in the anti-conformation relative

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to the sugars. Sugar-phosphate backbone and glycosidic torsion (Supplementary Table S1, Supporting Information) angles fall mostly in the typical B-DNA ranges; that is, the torsion angles R, β, γ, and δ are in their usual (-)-gauche, trans, (þ)-gauche, and (þ)-anticlinal ranges, respectively. The torsion angles R and γ show the largest deviations from this usual range (see Supporting Information). On the basis of torsion angles ε and ζ, the phosphate backbone adopts the BI conformation.39 The average glycosidic torsion angle of the undecamer is -104.3°, indicating an anti position of the bases according to the sugars. The sugar puckering modes are typical for a C20 -endo conformation, indicating the B-type character of the double helix. Deviations are related to the triplex formation and are further discussed in Section 3.2. Although the octamer parts of the undecamer d(TGGCCTTAAGG), the decamer d(GGCCAATTGG), and the nonamer d(GCGAATTCG) have similar intra and inter base pair parameters (see Supplementary Figures S1 and S2, Supporting Information), large fluctuations, especially in the TTAA sequence, exist. They are linked to the interaction of the third overhanging thymine bases with the central TTAA sequence, resulting in a more pronounced influence of the triplet formation on the twist angle value of the steps between the duplex and the antiparallel triplex region (see Supporting Information). 3.2. Base Triplets. The Watson-Crick parts of the base triplets are very similar to the normal Watson-Crick base pairs. The hydrogen-bond distances in the four G 3 GC base triplets are summarized in Table 2. In the next paragraphs Watson-Crick bases are denoted as GWC, CWC, AWC, TWC, while (reverse-)Hoogsteen bases are denoted as GH and TH. The geometry of both T 3 AT triplets and triple helical fragments consisting of three triplets is further described in the Supporting Information. Figure 3 shows the final 2Fo - Fc electron density map superposed on the triplets, formed in the reported structure of d(TGGCCTTAAGG). 3.3. Stacking. Besides the fact that the undecamer makes it possible to visualize the X-ray diffraction structure of a T 3 AT triplet, a second feature of the undecamer is the formation of two triple helical trimers by the interaction of the asymmetric unit with three other symmetry-equivalent strands (Figure 1), containing two T 3 AT triplets and four G 3 GC triplets. Here, one can show five triplet-to-triplet base stacking interactions instead of three in the decamer structure. Two of them present the interaction between a G 3 GC and a T 3 AT triplet. Except for the two central base pairs T6A27 and A7T26 (see Supplementary Figure S3, Supporting Information), all other bases are involved in the triplet formation. When comparing the stacking of the two T 3 AT triplets, one can observe a much better stacking of the Hoogsteen site, that is, T20 on G21. At the reverse-Hoogsteen site, a destacking occurs between T0 and G2, due to the 270° swung out thymine base, which searches for the best available space in the crystal packing when trying to optimize triplet interaction. Probably, as a consequence of this, a subsequent destacking appears for the guanines G1 and G2 (Figure S3, Supporting Information). The two trimers are stacked end-to-end on top of each other. The first three triplets form an antiparallel reverseHoogsteen d(T)d(GG) 3 d(AGG)d(TCC) triple helical trimer,


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Table 2. Watson-Crick and (reverse)-Hoogsteen Hydrogen Bonds for the Four G 3 GC and Two T 3 AT Triplets in the Undecamer d(TGGCCTTAAGG)a triplet Antiparallel G1H 3 G10WCC23WC

G2H 3 G9WCC24WC

T0H 3 A8WCT25WC

Parallel G21H 3 G29WCC4WC

hydrogen bond Watson-Crick

2.84 2.95 3.02 2.71 2.83 3.35 3.00 2.80 3.22 2.89 2.82 2.89 3.43 2.78

Watson-Crick

2.97 2.86 2.86 3.42 2.94 2.90 2.85 2.97 2.83 2.78 2.76 3.00 2.91 3.30 2.83 3.28

Watson-Crick

Hoogsteen

T20H 3 A28WCT5WC

distance (A˚)

N2(G10) 3 3 3 O2(C23) N1(G10) 3 3 3 N3(C23) O6(G10) 3 3 3 N4(C23) Reverse Hoogsteen N2(G1) 3 3 3 O6(G10) N1(G1) 3 3 3 N7(G10) Watson-Crick N2(G9) 3 3 3 O2(C24) N1(G9) 3 3 3 N3(C24) O6(G9) 3 3 3 N4(C24) Reverse Hoogsteen N2(G2) 3 3 3 O6(G9) N1(G2) 3 3 3 N7(G9) Watson-Crick N1(A8) 3 3 3 N3(T25) N6(A8) 3 3 3 O4(T25) Reverse Hoogsteen O2(T0) 3 3 3 N6(A8) N3(T0) 3 3 3 N7(A8)

Hoogsteen

G22H 3 G30WCC3WC

atoms

Watson-Crick Hoogsteen

N2(G29) 3 3 3 O2(C4) N1(G29) 3 3 3 N3(C4) O6(G29) 3 3 3 N4(C4) O6(G21) 3 3 3 N4(C4) N1(G21) 3 3 3 O6(G29) N2(G21) 3 3 3 N7(G29) N2(G30) 3 3 3 O2(C3) N1(G30) 3 3 3 N3(C3) O6(G30) 3 3 3 N4(C3) O6(G22) 3 3 3 N4(C3) N1(G22) 3 3 3 O6(G30) N2(G22) 3 3 3 N7(G30) N1(A28) 3 3 3 N3(T5) N6(A28) 3 3 3 O4(T5) O4(T20) 3 3 3 N6(A28) N3(T20) 3 3 3 N7(A28)

a Watson-Crick bases are denoted as CWC, GWC, TWC, and AWC, while (reverse-)Hoogsteen bases are denoted as GH and TH. G1H 3 G10WCC23WC, G2H 3 G9WCC24WC, and T0H 3 A8WCT25WC are antiparallel and G21H 3 G29WCC4WC, G22H 3 G30WCC3WC, and T20H 3 A28WCT5WC are parallel triplets.

whereas the last three interact in a parallel Hoogsteen mode forming a d(GGT) 3 d(GGA)d(CCT) triplet trimer (Figure 4). 3.4. Crystal Packing. The packing arrangement in the undecamer structure can be compared with the nonamer and decamer structures.22,24 Comparison of the cell dimensions and space group of the nonamer, decamer, and undecamer would suggest a similar crystal packing for these three structures. However, a few differences can be observed. The packing of the undecamer is somewhat looser than observed for the nonamer and the decamer as illustrated by the mean volume per base pair (1825 A˚3 with respect to 1391 and 1563 A˚3, for the nonamer and decamer, respectively). As the columns of the helices are built up by octamer duplexes, these values are calculated for eight base pairs per duplex, with four duplexes per unit cell. This is certainly not the consequence of an unwinding of the double helix: the undecamer contains 10.2 residues per turn compared to 10.2 and 10.1 for the nonamer and the decamer, respectively. The extra T 3 AT triplet in the parallel triple helical fragments strengthens the end-to-end packing of the helices in one column resulting in almost perfect helical columns. The separation between these columns is however somewhat larger. The reason for this can be 2-fold. First, no Mg(H2O)62þ clusters bridging adjacent phosphate backbone

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Figure 3. Antiparallel base triplets G1 3 G10C23 (a), G2 3 G9C24 (b), and T0 3 A8T25 (c), and parallel base triplets G21 3 G29C4 (d), G22 3 G30C3 (e), and T20 3 A28T5 (f) in the undecamer d(TGGCCTTAAGG). Reverse-Hoogsteen and Hoogsteen triplets are in the left and right column, respectively. All base triplets are in stick representation; the final 2Fo - Fc Fourier map density at 1.5σ (cyan) and 2.5σ (blue) contour level is superposed on the base pairs.

strands are found as in the decamer structure; in addition, the lower resolution also accounts for this. Such clusters can indeed allow a closer approach of adjacent phosphate groups. Second, the overhanging bases T0, G1, and G2 swing further out compared to the decamer structure in order to find an optimal position and interaction with neighboring bases. Finally, this results in an increase of about 5 A˚ of the lattice parameter b. 3.5. Hydration and Thermal Parameters. Although X-ray diffraction techniques have the advantage of revealing the positions of tightly bound water molecules, the limiting factor of accurately finding these waters is the resolution. Therefore, the total of solvent molecules clearly defined in the undecamer is restricted to 44. Nine water molecules could

be placed in the minor groove, representing part of the “(extended) spine of hydration” as was already observed in the crystal structures of the nonamer and the decamer.22,24 The mean B-value for the DNA atoms in the reported structure is 50.3 A˚2, with the two DNA strands differing in thermal motion (strand A and strand B show a mean B-value of 46.2 and 54.5 A˚2, respectively.) 3.6. Modeling. Construction of a DNA triplex requires the twist and rise between successive base triplets. The reported structure provides part of these helical parameters, and in combination with docking and optimization calculations, has allowed the generation of models of both parallel and antiparallel (G 3 GC)2/T 3 AT triple helices (Figure 5).

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Figure 4. Cross-eyed stereo stick representation of the antiparallel reverse-Hoogsteen d(T)d(GG) 3 d(AGG)d(TCC) (upper) and the parallel Hoogsteen d(GGT) 3 d(GGA)d(CCT) (lower) triple helical trimer. The trimer duplexes are shown in green, whereas the third strands, positioned in the major groove, are shown in red. Note there is a discontinuity in the backbone of the third strand of the reverse-Hoogsteen d(T)d(GG) 3 d(AGG)d(TCC) trimer.

Figure 5. View into the major groove occupied by the third strand of the parallel (A) and antiparallel (B) [(G 3 GC)2/T 3 AT]4 triple helical models. Both Watson-Crick strands are in green and the third (reverse-)Hoogsteen strand is in red. Coordinates are available as Supporting Information.

The parallel [(G 3 GC)2/T 3 AT]4 triplex has an average helical twist of ∼37.1° and a rise of ∼3.2 A˚. The twist and rise of the subsequent T 3 AT/G 3 GC (∼43.8°; ∼3.5 A˚) and G 3 GC/G 3 GC (∼25.9°; ∼3.2 A˚) steps are established from the reported structure, whereas the initially unknown twist and rise of the G 3 GC/T 3 AT step were calculated (∼43.1°; ∼2.9 A˚) (see Section 2.4). The values for the G 3 GC/G 3 GC steps are in agreement with the ones obtained for the previously constructed parallel (G 3 GC)12 triplex model (∼26.8°; ∼3.7 A˚).23 For the antiparallel [(G 3 GC)2/T 3 AT]4 triplex, the average helical twist is ∼36.9° and the rise is ∼3.5 A˚. The T 3 AT/ G 3 GC shows a much smaller twist of ∼31.1° (rise ∼ 3.2 A˚) in comparison with the parallel triplex, which is compensated by a much larger G 3 GC/G 3 GC twist of ∼37.9° (rise ∼3.3 A˚).

The docked G 3 GC/T 3 AT shows the same twist of ∼43.1° (rise ∼ 4.1 A˚) as the parallel one. The values for the G 3 GC/ G 3 GC step are less in agreement with the ones obtained for the previously constructed antiparallel (G 3 GC)12 triplex model (∼29.5°; ∼3.5 A˚).23 The twist and rise of the reported antiparallel [(G 3 GC)2/ T 3 AT]4 triplex is in agreement with the previously determined antiparallel triple helical NMR-models, containing T 3 AT/G 3 GC base triplets (NDB code 134D),40 with an average G 3 GC/G 3 GC twist of 34.0° and rise of 3.4 A˚. However, the average T 3 AT/G 3 GC and G 3 GC/T 3 AT twists of 23.2° (rise 3.4 A˚) and 34.8° (rise 2.9 A˚), respectively, are somehow smaller in comparison with the reported antiparallel [(G 3 GC)2/T 3 AT]4 triplex.

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When considering only the third reverse-Hoogsteen strand, the T-G and G-T steps show a similar rise (∼3.1 and ∼3.2 A˚, respectively) and large twist (∼41 and ∼42°), whereas the twist and rise for the G-G steps (∼28°; ∼3.2 A˚) are similar to those observed for the duplex region of the (G 3 GC)12 triplex. In the analysis of the NMR-model,40 it has been reported that predominantly the T-G steps differ significantly by an increased helical rise and reduced twist. 4. Conclusion Crystal engineering techniques, by exploiting the ability of overhanging bases in the undecamer d(TGGCCTTAAGG), confirms that a carefully chosen DNA sequence can reveal interesting features of short sections of parallel triple helices. The elongation of the decamer d(GGCCAATTGG) with one extra thymine residue at both ends of the helix, results in the formation of a parallel Py 3 PuPy triplet. Not only has the nature of the first T 3 AT triplet been examined but also the stacking interactions with the two base pair long Pu 3 PuPy triplex could be analyzed. At the other end of the helix, two guanine residues swing out of the helix to form antiparallel Pu 3 PuPy triplets with GC base pairs of a neighboring duplex of an adjacent column. Because no space is left for an analogue’s interaction of the overhanging thymine residue, the position of the thymine was unexpected. It swings out over an extra 90° compared to the guanines to interact with an AT base pair of a third symmetry equivalent duplex. In the resulting antiparallel reverse Hoogsteen T 3 AT triplet, the thymine residue is more searching for space instead of optimizing the interaction with an AT base pair. Because the lack of any covalent bindings with the G2G1 bases, the thymine base has more degrees of freedom. This is illustrated by its almost fully stretched backbone. Crystal engineering techniques have already resulted in a successful crystallization of the nonamer d(GCGAATTCG), containing two G 3 CG triplets, the decamer d(GGCCAATTGG), containing four G 3 CG triplets, and finally the undecamer d(TGGCCTTAAGG), containing four G 3 CG and two T 3 AT triplets, resulting in the formation of a parallel and an antiparallel trimer. A further elongation with an extra thymine residue in the dodecamer d(TTGGCCTTAAGG) could give rise to the formation of a total of eight triplets. Until now the crystallization of the latter was unsuccessful, only leading to fiber-diffracting crystals. In the future, accurate screening of the crystallization conditions should finally lead to qualitatively good crystals and structure solution of triple helical fragments containing four triplet steps. Acknowledgment. We thank the staff of the EMBL Hamburg Outstation for their support with the synchrotron experiments. Support from the European Community - Research Infrastructure Action under the FP6 “Structuring the European Research Area Programme”, contract number: RII3-CT2004-506008 is gratefully acknowledged. BioMacS, the K.U.Leuven Interfacultary Centre for Biomacromolecular Structure, is supported by the Impulse Project of the K.U.Leuven. Supporting Information Available: Tables S1-S4 and coordinates for the parallel and antiparallel [(G 3 GC)2/T 3 AT]4 triple

Van Hecke et al. helical models are available free of charge via the Internet at http://pubs.acs.org.

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