James P. Hall

Reversal of a Single Base‐Pair Step Controls Guanine Photo‐Oxidation by an Intercalating Ruthenium(II) Dipyridophenazine Complex

Abstract Small changes in DNA sequence can often have major biological effects. Here the rates and yields of guanine photo‐oxidation by Λ‐[Ru(TAP)2(dppz)]2+ have been compared in 5′‐{CCGGATCCGG}2 and 5′‐{CCGGTACCGG}2 using pico/nanosecond transient visible and time‐resolved IR (TRIR) spectroscopy. The inefficiency of electron transfer in the TA sequence is consistent with the 5′‐TA‐3′ versus 5′‐AT‐3′ binding preference predicted by X‐ray crystallography. The TRIR spectra also reveal the differences in binding sites in the two oligonucleotides.

Delta chirality ruthenium 'light-switch' complexes can bind in the minor groove of DNA with five different binding modes.

[Ru(phen)2(dppz)]2+ has been studied since the 1990s due to its ‘light-switch’ properties. It can be used as a luminescent DNA probe, with emission switched on through DNA binding. The luminescence observed is dependent on the solvent accessibility of the pyrazine nitrogen atoms, and therefore is sensitive to changes in both binding site of the cation and chromophore orientation. The compound is also chiral, and there are distinct differences between the enantiomers in terms of the emission behaviour when bound to a variety of DNA sequences. Whilst a number of binary DNA-complex X-ray crystal structures are available, most include the Λ enantiomer and there is very little structural information about binding of the Δ enantiomer. Here, we present the first X-ray crystal structure of a Δ enantiomer bound to well-matched DNA, in the absence of the other, Λ enantiomer. We show how the binding site observed here can be related to a more general pattern of motifs in the crystallographic literature and propose that the Δ enantiomer can bind with five different binding modes, offering a new hypothesis for the interpretation of solution data.

Controlled Dehydration of a Ruthenium Complex-DNA Crystal Induces Reversible DNA Kinking.

Hydration-dependent DNA deformation has been known since Rosalind Franklin recognized that the relative humidity of the sample had to be maintained to observe a single conformation in DNA fiber diffraction. We now report for the first time the crystal structure, at the atomic level, of a dehydrated form of a DNA duplex and demonstrate the reversible interconversion to the hydrated form at room temperature. This system, containing d(TCGGCGCCGA) in the presence of Λ-[Ru(TAP)2(dppz)](2+) (TAP = 1,4,5,8-tetraazaphenanthrene, dppz = dipyrido[3,2-a:2,3-c]phenazine), undergoes a partial transition from an A/B hybrid to the A-DNA conformation, at 84-79% relative humidity. This is accompanied by an increase in kink at the central step from 22° to 51°, with a large movement of the terminal bases forming the intercalation site. This transition is reversible on rehydration. Seven data sets, collected from one crystal at room temperature, show the consequences of dehydration at near-atomic resolution. This result highlights that crystals, traditionally thought of as static systems, are still dynamic and therefore can be the subject of further experimentation.

Preferred orientation in an angled intercalation site of a chloro-substituted Λ-[Ru(TAP)2(dppz)]2+ complex bound to d(TCGGCGCCGA)2

The crystal structure of the ruthenium DNA ‘light-switch’ complex Λ-[Ru(TAP)2(11-Cl-dppz)]2+ (TAP=tetraazaphenanthrene, dppz=dipyrido[3,2-a′:2′,3′-c]phenazine) bound to the oligonucleotide duplex d(TCGGCGCCGA)2 is reported. The synthesis of the racemic ruthenium complex is described for the first time, and the racemate was used in this study. The crystal structure, at atomic resolution (1.0u2009Å), shows one ligand as a wedge in the minor groove, resulting in the 51° kinking of the double helix, as with the parent Λ-[Ru(TAP)2(dppz)]2+. Each complex binds to one duplex by intercalation of the dppz ligand and also by semi-intercalation of one of the orthogonal TAP ligands into a second symmetrically equivalent duplex. The 11-chloro substituent binds with the major component (66%) oriented with the 11-chloro substituent on the purine side of the terminal step of the duplex.

Monitoring guanine photo-oxidation by enantiomerically resolved Ru(II) dipyridophenazine complexes using inosine-substituted oligonucleotides.

The intercalating [Ru(TAP)2(dppz)](2+) complex can photo-oxidise guanine in DNA, although in mixed-sequence DNA it can be difficult to understand the precise mechanism due to uncertainties in where and how the complex is bound. Replacement of guanine with the less oxidisable inosine (I) base can be used to understand the mechanism of electron transfer (ET). Here the ET has been compared for both Λ- and Δ-enantiomers of [Ru(TAP)2(dppz)](2+) in a set of sequences where guanines in the readily oxidisable GG step in {TCGGCGCCGA}2 have been replaced with I. The ET has been monitored using picosecond and nanosecond transient absorption and picosecond time-resolved IR spectroscopy. In both cases inosine replacement leads to a diminished yield, but the trends are strikingly different for Λ- and Δ-complexes.

Guanine Can Direct Binding Specificity of Ru–dipyridophenazine (dppz) Complexes to DNA through Steric Effects

Abstract X‐ray crystal structures of three Λ‐[Ru(L)2dppz]2+ complexes (dppz=dipyridophenazine; L=1,10‐phenanthroline (phen), 2,2′‐bipyridine (bpy)) bound to d((5BrC)GGC/GCCG) showed the compounds intercalated at a 5′‐CG‐3′ step. The compounds bind through canted intercalation, with the binding angle determined by the guanine NH2 group, in contrast to symmetrical intercalation previously observed at 5′‐TA‐3′ sites. This result suggests that canted intercalation is preferred at 5′‐CG‐3′ sites even though the site itself is symmetrical, and we hypothesise that symmetrical intercalation in a 5′‐CG‐3′ step could give rise to a longer luminescence lifetime than canted intercalation.

Inosine can increase DNA's susceptibility to photo-oxidation by a Ru(II) complex due to structural change in the minor groove

Key to the development of DNA-targeting phototherapeutic drugs is determining the interplay between the photoactivity of the drug and its binding preference for a target sequence. For the photo-oxidising lambda-[Ru(TAP)2 (dppz)]2+ (Λ-1) (dppz=dipyridophenazine) complex bound to either d{T1 C2 G3 G4 C5 G6 C7 C8 G9 A10 }2 (G9) or d{TCGGCGCCIA}2 (I9), the X-ray crystal structures show the dppz intercalated at the terminal T1 C2 ;G9 A10 step or T1 C2 ;I9 A10 step. Thus substitution of the G9 nucleobase by inosine does not affect intercalation in the solid state although with I9 the dppz is more deeply inserted. In solution it is found that the extent of guanine photo-oxidation, and the rate of back electron-transfer, as determined by pico- and nanosecond time-resolved infrared and transient visible absorption spectroscopy, is enhanced in I9, despite it containing the less oxidisable inosine. This is attributed to the nature of the binding in the minor groove due to the absence of an NH2 group. Similar behaviour and the same binding site in the crystal are found for d{TTGGCGCCAA}2 (A9). In solution, we propose that intercalation occurs at the C2 G3 ;C8 I9 or T2 G3 ;C8 A9 steps, respectively, with G3 the likely target for photo-oxidation. This demonstrates how changes in the minor groove (in this case removal of an NH2 group) can facilitate binding of RuII dppz complexes and hence influence any sensitised reactions occurring at these sites. No similar enhancement of photooxidation on binding to I9 is found for the delta enantiomer.

Chapter 8:Structural Studies of DNA-binding Metal Complexes of Therapeutic Importance

Ruthenium polypyridyl complexes are of interest for their possible applications as cellular probes, in anticancer therapeutics and, most recently, for their antibacterial properties. For many years there was no crystallographic evidence showing how any of these complexes bound to duplex or higher-order DNA, but since 2011 a series of structural studies have shown aspects of sequence, enantiomeric, substituent and structural specificity. The principal binding mode to duplex DNA of complexes typified by [Ru(phen)2dppz]2+ (where dppz=dipyridophenazene) is by angled (canted) intercalation from the minor groove, with a distinct symmetric binding mode so far only known for lambda enantiomers at the TA/TA steps. Kinking (semi-intercalation) has also been characterised, so far only at CC/GG steps, e.g. for phen ligands within these complexes. Delta enantiomers are capable of mismatch recognition, so far structurally characterised for the A–A mismatch. This binding mode, insertion, is characterised by the flipping out of the adenine, with the base stacking on the ancillary ligand of the complex. For binding to higher-order DNA, sequences with loops, such as the unimolecular G-quadruplex, have so far resisted attempts at crystallisation, although an NMR structure of a diruthenium complex has been reported.

X-ray crystal structures show DNA stacking advantage of terminal nitrile substitution in Ru-dppz complexes

The new complexes [Ru(TAP)2 (11-CN-dppz)]2+ , [Ru(TAP)2 (11-Br-dppz)]2+ and [Ru(TAP)2 (11,12-diCN-dppz)]2+ are reported. The addition of nitrile substituents to the dppz ligand of the DNA photo-oxidising complex [Ru(TAP)2 (dppz)]2+ promote π-stacking interactions and ordered binding to DNA, as shown by X-ray crystallography. The structure of Λ-[Ru(TAP)2 (11-CN-dppz)]2+ with the DNA duplex d(TCGGCGCCGA)2 shows, for the first time with this class of complex, a closed intercalation cavity with an AT base pair at the terminus. The structure obtained is compared to that formed with the 11-Br and 11,12-dinitrile derivatives, highlighting the stabilization of syn guanine by this enantiomer when the terminal base pair is GC. In contrast the AT base pair has the normal Watson-Crick orientation, highlighting the difference in charge distribution between the two purine bases and the complementarity of the dppz-purine interaction. The asymmetry of the cavity highlights the importance of the purine-dppz-purine stacking interaction.