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-a midine element related to the antibiotic netropsin is covalently tethered t o 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 hybri d molecule binds to AT-rich sequences of DNA via a bimodal process involvin g minor groove binding of the netropsin moiety and intercalation of the phe nazine moiety. The bidentate mode of binding was evidenced by Linear dichro ism using calf thymus DNA and poly(dA-dT).(dA-dT). In contrast, the drug fa ils 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 polynucleo tide. DNase I footprinting studies indicated that the conjugate interacts p referentially with AT-rich sequences, but the cleavage of DNA in the presen ce of a reducing agent can occur at different sequences not restricted to t he AT sites. The main cleavage sites were detected with a periodicity of ab out 10 base pairs corresponding to approximately one turn of the double hel ix. This suggests that the cleavage may be dictated by the structure of the double helix rather than the primary nucleotide sequence. The conjugate wh ich is moderately toxic to cancer cells complements the tool box of reagent s which can be utilized to produce DNA strand scission. The DNA cleaving pr operties of R-128 entreat further exploration into the use of phenazine-di- N-oxides as tools for investigating DNA structure.