3.3 Results and discussion
3.3.4 Differences and similarities between the two structures
case, we suspect that the preference of the metal complex for the minor groove may be a result of the major groove being less available in the crystal. In this structure, two large densities are present in the major groove, which are tentatively fit to Ba atoms, coordinated by the O6 and N7 of guanines. A Ba atom was also located in the major groove in a recent structure of Λ-Ru(TAP)2dppz2+ bound to DNA.7 One of the Ba atoms is only one base pair away from the 5’-A9C10-3’ intercalation site. At the same time, the ruthenium complex at 5’-A6T7-3’, intercalated deeply into the DNA base stack, has its dppz ligand protruding out into the major groove. Thus, the space in the major groove becomes rather restricted, which may have prompted the last ruthenium complex to bind in the minor groove even without ancillary interactions.
In this crystal, stacking from extruded thymidines connects two side-by-side, antiparal- lel, crystallographically related duplexes (Figure 3.9 and 3.10). Symmetry-related duplexes are also stacked head-to-tail to form a rod along the long axis (Figure 3.10). The two rods stemming from the two side-by-side duplexes are at an angle of ∼35◦ with respect to each other (Figure 3.10). Thus, each duplex within a rod forms thymine-thymine interactions with a different rod in the lattice. Altogether, parallel rods and their thymine-stacking partner rods make up a crisscross pattern in the lattice.
Figure 3.9. Two side-by-side, crystallographically related duplexes are connected through thymine-thymine (red) stacking.
Figure 3.10. Long rods formed by end-to-end stacking of symmetry-related duplexes are positioned at an angle with respect to each other.
Taken together, the two structures offered three independent views of metalloinsertion, five of metallointercalation, and two of end-capping. All binding events occurred from the minor groove, with the dppz ligand inserted deeply into the DNA base stack,π-stacking with the bases above and below the binding site. The orientation of the dppz ligand varies quite significantly among the binding sites. In structure 1, the dppz ligand is always centered between the sugar-phosphate backbones, but in structure 2, it is always intercalated side- ways (Figure 3.11). Given that dppz is a narrow but long ligand with the entire phenazine portion available for π-π stacking, we had previously proposed these two types of binding orientation based on NMR and luminescence lifetime studies.6,29 Although our proposal was predicated on binding in the major groove, the crystal structures presented here clearly show that even when the ruthenium complex is restricted in the minor groove, it can bind in either a head-on or canted fashion. In the head-on orientation, the two phenazine nitrogens are equally protected from the solvent; in the canted orientation, one nitrogen is perceiv- ably more solvent accessible than the other. The difference in binding orientation in the crystal structures may have originated from the DNA being more “symmetric” in structure 1but less so in structure2. In the former, the two symmetric AA mismatches are the most destabilized regions of the duplex, where initial binding of ruthenium by metalloinsertion must have occurred. Curiously, in the second crystal, even though the DNA sequence is also palindromic, it appears that once the ruthenium recognizes a weak point (the extruded AT) in the helix, the rest of the helix must have been affected in a way that the previously symmetric AT pair is no longer a weak point (in the case of an AA mismatch, it will al- ways remain destabilized relative to well-matched regions). Consequently, the remaining ruthenium complexes are intercalated along the helix at different steps from those in the mismatched duplex. Furthermore, the two AT pairs are only two base pairs away from each other (versus four base-pair separation for the AA mismatches), and it is possible that this difference ultimately affects binding geometries.
Besides intercalation through the dppz ligand, the most striking similarity between the two structures is the prevailing ancillary interactions between two bpy ligands, or a bpy and an extruded base. Ancillary interactions are present in related rhodium-DNA structures, but this is the first time we observe direct stacking between two neighboring complexes, or between an extruded base and the very complex that has caused its ejection. In two of the rhodium-DNA structures, a Rh(bpy)2chrysi3+ is intercalated between well-matched base
Figure 3.11. Ruthenium intercalation in structure2at 5-’G2G3-3’ (top), 5’-A6T7-3’ (mid- dle) and 5’-A9C10-3’ (bottom).
Table 3.3. DNA base step parameters in structure2a
Step Ru binding mode Shift (˚A) Slide (˚A) Rise (˚A) Tilt (◦) Roll (◦) Twist (◦)
C1/G2 – -0.04 0.88 2.91 -1.06 -0.76 27.48
G2/G3 intercalation -0.13 0.52 7.01 -4.16 15.99 26.76
G3/T4 – -0.57 -0.02 2.91 -1.90 0.66 21.47
T4/A6 insertion 0.42 4.60 6.83 -5.95 1.05 80.41
A6/T7 intercalation 0.78 -0.07 6.43 3.24 13.50 8.44
T7/T8 – -0.10 0.56 3.19 3.23 4.60 32.20
T8/A9 – -0.53 1.74 3.25 1.63 5.74 34.38
A9/C10 intercalation -0.22 0.18 6.65 -19.15 13.88 22.07
C10/C11 – -0.03 -0.10 3.03 5.22 -0.72 25.37
B-DNA – -0.1 -0.8 3.3 -1.3 -3.6 36
aData were calculated using 3DNA.34
pairs from the major groove, while its bpy ligands are end-stacked with crystallographically related duplexes.16,19The intercalative Rh binding event, absent in solution, was attributed to crystal packing through ancillary interactions. Likewise, in the two ruthenium-DNA structures, these interactions act to stabilize intercalated ruthenium complexes, rigidifying the entire structure and potentially facilitating crystallization.
Overall, the DNA helix in both structures is unwound at almost every step – the majority of the helical twist parameter between two consecutive, stacked base pairs is between 20 and 30◦ – so as to accommodate the high density of intercalated metal complexes (Table 3.2 and 3.3). Both structures also show a wide range of propeller and buckle angles, as the DNA adapts to multiple ligand binding and base extrusion. All of the sugar pucker is C2’-endo or the closely-related C3’-exo and C1’-exo. Given that five independent views of intercalation are illustrated between the two structures, it seems that alternating C3’-endo/C2’-endo puckering is not a requirement for intercalation.