A Reinvestigation by Circular Dichroism and NMR:  Ruthenium(II) and Rhodium(III) Metallointercalators Do Not Bind Cooperatively to DNA

Fast, long-range electron transfer mediated by the DNA helix has been questioned by some researchers citing the possible clustering, or cooperative association, of noncovalently bound donors and acceptors on DNA. A systematic investigation of binding to DNA by the metallointercalators Δ-bis(1,10-phenanthroline)(dipyridophenazine)ruthenium(II) (Ru), Δ-bis(9,10-phenanthrenequinone diimine)(2,2‘-bipyridine)rhodium(III) (Rh), and Δ-bis(9, 10-phenanthrenequinone diimine)(5-(amidoglutaryl)-1,10-phenanthroline)rhodium(III) (Rh‘) using circular dichroism and NMR has shown no evidence for their cooperative clustering on a DNA helix. Circular dichroism (CD) studies of Ru and Rh in [poly(dA-dT)]_2, mixed-sequence calf thymus DNA, and [poly(dG-dC)]_2 as a function of loading indicate that the largest perturbations to the CD signal occur upon initial addition of DNA, with no subsequent systematic variation. Difference spectra of the two metallointercalators bound together versus separately are virtually indistinguishable at high and low loadings. Two-dimensional 1H NMR studies of Ru and Rh‘ binding to a DNA decamer duplex in 90:10 H_2O/D_2O have also been conducted. A more direct structural picture of the site occupancies of these complexes on a DNA helix emerges through examination of the dramatic upfield shifts of the imino protons of the DNA bases that occur upon intercalation. These studies reveal that with both complexes present, each intercalates specifically toward either end of the duplex, with a 4 base pair separation between them. In contrast, the complexes individually bound to the duplex showed low site-selectivity, and preferred more central sites. If anything, these data indicate anti-cooperative binding to the helix, which might be expected based upon electrostatic considerations. Time-resolved measurements of the Ru(II) luminescence reveal substantial subnanosecond quenching (approximately 60%) in the presence of Rh(III). Based upon the NMR results, this quenching must proceed over a distance >14 Å via electron transfer through the DNA π-stack. These experiments with noncovalently bound intercalators are fully consistent with earlier studies of electron transfer through DNA utilizing covalently bound donors and acceptors and definitively prove clustering cannot be responsible for the fast photoinduced electron transfer between metallointercalators mediated by the DNA double helix