Sequence-recognition and cleavage of DNA by a netropsin-phenazine-di-N-oxide conjugate

We report the synthesis, DNA-binding and cleaving properties, and cytotoxic activities of R-128, a hybrid molecule in which a bis-pyrrolecarboxamide-amidine element related to the antibiotic netropsin is covalently tethered to a phenazine-di-N-oxide chromophore. The affinity and mode of interaction of the conjugate with DNA were investigated by a combination of absorption spectroscopy, circular dichroism, and electric linear dichroism. This hybrid molecule binds to AT-rich sequences of DNA via a bimodal process involving minor groove binding of the netropsin moiety and intercalation of the phenazine moiety. The bidentate mode of binding was evidenced by linear dichroism using calf thymus DNA and poly(dA-dT).(dA-dT). In contrast, the drug fails to bind to poly(dG-dC).poly(dG-dC), because of the obstructive effect of the guanine 2-amino group exposed in the minor groove of this polynucleotide. DNase I footprinting studies indicated that the conjugate interacts preferentially with AT-rich sequences, but the cleavage of DNA in the presence of a reducing agent can occur at different sequences not restricted to the AT sites. The main cleavage sites were detected with a periodicity of about 10 base pairs corresponding to approximately one turn of the double helix. This suggests that the cleavage may be dictated by the structure of the double helix rather than the primary nucleotide sequence. The conjugate which is moderately toxic to cancer cells complements the tool box of reagents which can be utilized to produce DNA strand scission. The DNA cleaving properties of R-128 entreat further exploration into the use of phenazine-di-N-oxides as tools for investigating DNA structure.