John A. Brazier

X‑ray crystal structure of rac-[Ru(phen)2dppz]2+ with d(ATGCAT)2 shows enantiomer orientations and water ordering

We report an atomic resolution X-ray crystal structure containing both enantiomers of rac-[Ru(phen)2dppz](2+) with the d(ATGCAT)2 DNA duplex (phen = phenanthroline; dppz = dipyridophenazine). The first example of any enantiomeric pair crystallized with a DNA duplex shows different orientations of the Λ and Δ binding sites, separated by a clearly defined structured water monolayer. Job plots show that the same species is present in solution. Each enantiomer is bound at a TG/CA step and shows intercalation from the minor groove. One water molecule is directly located on one phenazine N atom in the Δ-enantiomer only.

Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy

To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ultrafast time-resolved infrared (TRIR) spectroscopy was performed on photoexcited ruthenium polypyridyl-DNA crystals, the atomic structure of which was determined by X-ray crystallography. By combining the X-ray and TRIR data we are able to define both the geometry of the reaction site and the rates of individual steps in a reversible photoinduced electron-transfer process. This allows us to propose an individual guanine as the reaction site and, intriguingly, reveals that the dynamics in the crystal state are quite similar to those observed in the solvent medium.

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.

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase

Significance Alterations in nonduplex structures could play roles during DNA replication in the progression of cancer and other intractable diseases. These noncanonical structures differ topologically from each other. However, the role of these differences in diseases remains unknown. In this study, we found that the presence of i-motif structures in the template caused the DNA polymerase to stall immediately before these structures. The i-motif structures are more efficient than other structures, such as G-quadruplexes and hairpins, although their thermodynamic stabilities are similar. This inhibition effect of the DNA polymerase was regulated by molecular crowding, which mimics conditions in the cell. Therefore, it is possible that the i-motif could impede DNA replication or repair and cause genomic instability. Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases.

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.

Synthesis of 3′- S-Phosphorothiolate Oligonucleotides for their Potential Use in RNA Interference

The potency of RNA interference (RNAi) undoubtedly can be improved through chemical modifications to the small interfering RNAs (siRNA). By incorporation of the 3′-S-phosphorothiolate modification into strands of RNA, it is hoped that specific regions of a siRNA duplex can be stabilised to enhance the target binding affinity of a selected antisense strand into the activated RNA-induced silencing complex (RISC*). Oligonucleotides composed entirely of this modification are desirable so unconventional 5′ → 3′ synthesis is investigated, with initial solution-phase testing proving successful. The phosphoroamidite monomer required for solid-phase synthesis has also been produced.

Stabilization Of Multi-Stranded Nucleic Acid Structures Using 3″-S-Phosphorothiolate Linkages

3 ′-S-Phosphorothiolate linkages incorporated into an oligodeoxynucleotide have been shown to stabilise duplex formation with a complementary RNA strand, but destabilise a duplex formed with a complementary DNA strand. The four-stranded i-motif structure is also stabilised this modification.

Long‐Lived Excited‐State Dynamics of i‐Motif Structures Probed by Time‐Resolved Infrared Spectroscopy

UV-generated excited states of cytosine (C) nucleobases are precursors to mutagenic photoproduct formation. The i-motif formed from C-rich sequences is known to exhibit high yields of long-lived excited states following UV absorption. Here the excited states of several i-motif structures have been characterized following 267 nm laser excitation using time-resolved infrared spectroscopy (TRIR). All structures possess a long-lived excited state of ∼300 ps and notably in some cases decays greater than 1 ns are observed. These unusually long-lived lifetimes are attributed to the interdigitated DNA structure which prevents direct base stacking overlap.

Strong positive cooperativity in binding to the A3T3 repeat by Hoechst 33258 derivatives attaching the quinoline units at the end of a branched linker.

Hoechst 33258 derivatives with additional interacting moieties attached at the ends of branched linkers were synthesized, and their DNA binding properties were investigated with regard to the A3T3 repeat by measuring fluorescence spectra. The binding property of the ligand was investigated by fluorescence titration, and the titration data were analyzed using the McGhee-von Hippel method. Ligand 6Q with the quinolin-6-yloxyacetyl group and Ligand IQ with isoquinolin-6-yloxyacetyl group at the ends of the branched linkers exhibit highly positive cooperativity for the DNA having 5 A3T3 sites with 3 base-insertions between them with sequence selectivity. The strategy developed in this study may be generally applicable for designing ligands for repetitive DNA sequences.

CONTROL OF DNA CONFORMATION USING 3′-S-PHOSPHOROTHIOLATE-MODIFIED LINKAGES

An in-depth study into the incorporation of multiple 3′-S-phosphorothiolate modifications into oligodeoxynucleotides (ODNs) and their subsequent effect on ODN/DNA and ODN/RNA duplex stability. 3′-S-Phosphorothiolate linkages increase the stability of ODN/RNA duplexes and decrease the stability of ODN/DNA duplexes.