meso-[{Ru(phen)2}2(µ-HAT)]4+: a high-affinity DNA hairpin probe {HAT = 1,4,5,8,9,12-hexaazatriphenylene; phen = 1,10-phenanthroline}

Abstract

¹ H NMR spectroscopy and fluorescent intercalator displacement (FID) assays have been used to investigate the DNA-binding abilities of two series of dinuclear polypyridyl ruthenium(II) complexes of the form [{Ru(L)₂}₂(µ-BL)]⁴⁺ {L = 2,2′-bipyridine (bpy), 4,4′-dimethyl-2,2′-bipyridine (Me₂bpy), 1,10-phenanthroline (phen), or 4,7-dimethyl-1,10-phenanthroline (Me₂phen); BL = 2,2′-bipyrimidine (bpm) or 1,4,5,8,9,12-hexaazatriphenylene (HAT)}. Preliminary FID surveys of these metal complexes against a variety of different oligonucleotides revealed that those complexes based upon the HAT bridging ligand induced greater fluorescence decreases in dye-bound DNA than did their bpm-bridged counterparts, suggesting a higher binding affinity by the HAT-bridged species. Furthermore, the greatest fluorescence decreases were typically observed in an oligonucleotide featuring a six-base hairpin loop. The apparent binding affinity of the metal complexes was also found to be a function of the stereochemistry and identity of the terminal ligands of the complex. The meso (ΔΛ) stereoisomer generally induced greater fluorescence decreases than did either enantiomer (ΔΔ or ΛΛ), phen-based terminal ligands performed better than bpy-based terminal ligands, and those terminal ligands with methyl substituents demonstrated stronger apparent binding than did their non-methylated analogues. NMR experiments on meso-[{Ru(phen)₂}₂(µ-HAT)]⁴⁺ and meso-[{Ru(Me₂phen)₂}₂(µ-HAT)]⁴⁺ demonstrated that both complexes bound with high affinity to the six-base hairpin oligonucleotide at the stem–loop interface and provided evidence to support stronger binding by the methylated species. meso-[{Ru(phen)₂}₂(µ-HAT)]⁴⁺ was found to bind poorly to duplex DNA and smaller four-base hairpin loops in FID and NMR experiments, whereas FID data suggest that the methylated analogue binds relatively strongly to most oligonucleotide sequences (the four- and six-base hairpins in particular). These results demonstrate that binding affinity can come at the expense of selectivity, with meso-[{Ru(phen)₂}₂(µ-HAT)]⁴⁺ proving to be an efficient compromise between the two as a high-affinity DNA hairpin probe.