An approach to therapeutic agents through selective targeting of destabilised nucleic acid duplex sequences

Abstract

The binding of ΔΔ/ΛΛ-[{Ru(phen)₂}₂(μ-bb_n)]⁴⁺ {where phen = 1,10-phenanthroline, bb_n = 1,n-bis[4(4′-methyl-2,2′-bipyridyl)]-alkane (ΔΔ/ΛΛ-Rubb_n)} to the non-self complementary oligonucleotide 5′-d(CGCGATAAGCCGC·5′-GCGGCATTACGCG) (3-DB) has been examined using a 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) displacement assay. The 3-DB oligonucleotide contains two single adenine bulge nucleotides that are separated by three base pairs. ¹H NMR spectroscopy data demonstrated that the adenine bases are intra-helical and that the segment containing the two bulge nucleotides and the three A·T base pairs between the bulges forms a destabilised segment within the stable duplex oligonucleotide. The DAPI displacement assay demonstrated that ΔΔ-Rubb₇-bound 3-DB with higher affinity than the other members of the ΔΔ/ΛΛ-Rubb_n series. Molecular models suggested that the seven-carbon chain length in ΔΔ-Rubb₇ was ideal to span the distance between the two bulge sites. The binding of ΔΔ-Rubb₇ to 3-DB was also studied by ¹H NMR spectroscopy and molecular modelling. The selective changes in chemical shifts for the resonances from 3-DB upon addition of ΔΔ-Rubb₇ suggested that the metal complex specifically bound at the destabilised segment between A₅ and A₁₉. Observation in NOESY spectra of NOE cross peaks between 3-DB and ΔΔ-Rubb₇ confirmed that one of the ruthenium centres bound at the A₅ bulge site, with the other metal centre positioned at the A₁₉ bulge. In addition, ΔΔ-Rubb₇ was found to bind chromosomal DNA extracted from a suspension of Staphylococcus aureus that had been incubated with the ruthenium(II) complex. As inert dinuclear ruthenium(II) complexes are capable of being transported into a bacterial cell and bind chromosomal DNA, it is possible that they could be developed into anti-microbial agents that specifically target destabilised segments of DNA that are recognised by essential DNA-binding proteins.