Dipyridophenazine Complexes of Ruthenium(II) as Luminescent Reporters of DNA

Abstract

The search for novel diagnostics for DNA detection has generated interest in the potential applications of polypyridyl complexes of ruthenium(II). Recently, it was reported that dipyridophenazine complexes of ruthenium(II) may serve as molecular light switches for DNA. These complexes, which bind DNA avidly through intercalation, show no luminescence in aqueous solutions. Upon intercalation into double-helical DNA and the concomitant protection of the phenazine ring from quenching by interaction with water, intense photoluminescence is apparent. The light switch effect as a function of nucleic acid sequence and conformation was examined for [Ru(phen)2dppz]2+ and [Ru(bpy)2dppz]2+. The emission properties of these complexes were found to be extremely sensitive to the nature of the intercalation environment with strong correlations between the luminescence parameters and the level of water protection afforded by the double helix. In order to impart sequence specificity to the light switch effect, various methods have been developed for appending a functionalized [Ru(phen)2dppz]2+ to the 5' terminus of oligonucleotides, both on the solid support and in solution. Assays for analyzing the structural integrity of the resulting conjugates are described. These ruthenated oligonucleotides can serve as enzyme substrates, enabling the construction of long metalated oligonucleotides not easily prepared using chemical synthesis. In order to evaluate their utility as useful DNA diagnostics, a series of ruthenated oligonucleotides were synthesized and their photophysical properties characterized. Biochemical analysis of oligonucleotide duplexes containing ruthenated strands showed no significant structural perturbation of the duplex as a result of the ruthenium modification. The overall results of this investigation suggest that an oligonucleotide functionalized with a dppz complex of ruthenium may be used to target single-stranded DNA in a sequence-specific fashion and that this derivative could be extremely valuable in the development of novel hybridization probes for both heterogeneous and homogeneous assays.