Kinetic Recognition of Nucleic Acids - Studies on the DNA Binding Selectivity of Threading Ruthenium Complexes

Doctoral thesis, 2009

Despite the great progress in our understanding of DNA during the past half-century, there are many important aspects of its chemical and biological role yet to be explored. The principles by which it selectively interacts with other molecules have attracted much interest due to the relevance for fundamental cellular processes, as well as for the development of diagnostic probes and effective pharmaceutical agents. This Thesis describes the study of the process in which a planar aromatic moiety, hindered by bulky substituents on both ends, is threaded through the DNA double helix. Dumb-bell shaped binuclear ruthenium complexes of the type [µ-(bidppz)(L)4Ru2]4+, L = phenanthroline (P) or bipyridine (B) bind upon mixing with DNA rapidly on the outside of the double-helix, after which they rearrange to an intercalated binding mode. Passing one large metal centre between the strands requires large transient distortions of the duplex, leading to extremely slow binding kinetics that is sensitive to DNA sequence as well as ruthenium complex structure. This work has (1) addressed the mechanisms behind this “kinetic recognition” and (2) identified potential DNA structural targets. Both enantiomers of chiral analogues P and B require several hours at 50°C to rearrange to the threaded binding mode in mixed sequence DNA. Alternating AT polymers, on the other hand, are intercalated within a few minutes at room temperature. The ratio between the forward rates is estimated to vary between 65 (ΛΛ-P) and 2500 (ΛΛ-B). Studies with AT-tract oligonucleotides show that more than one complete helix turn of AT-DNA is required for efficient threading, a stretch considerably larger than the complexes themselves. Long AT-stretches are however not the only kinetically favored targets; subjecting mixed sequence DNA to negative supercoiling can increase the threading rate by as much as two orders of magnitude. Accelerated intercalation is also observed with partially unpaired DNA. Dissociation from mixed sequence DNA displays half-lives of up to 38 h at physiological temperature, the slowest release reported for a reversibly bound agent. The selectivity demonstrated by the binuclear ruthenium complexes in vitro make them interesting in the development of new agents against parasitic protozoa with AT-rich DNA.