Â© The Royal Society of Chemistry 2015. The synthesis, spectroscopic characterisation and biological evaluation of mono- and bis-1,8-naphthalimide-conjugated ruthenium(ii)-polypyridyl complexes is presented. Spectroscopic DNA titrations, together with denaturation studies, show strong binding of both species to DNA through the naphthalimide arms. Linear and circular dichroism (LD and CD) spectroscopy reveal close association of the Ru(bpy)32+ core with DNA in the case of the mono-naphthalamide complex, [Ru(bpy)2(bpy-NAP)]2+. Significantly, binding by the second naphthalimide arm in the [Ru(bpy)2(bpy-NAP2)]2+ complex is found to displace the Ru(bpy)32+ centre from the DNA backbone. This 'negative allosteric effect' is found to have a dramatic influence on the photoinduced damage of plasmid DNA, and the viability of HeLa cancer cells upon photoactivation. Overall the study clearly maps and correlates the relationship between molecular structure, in vitro binding and activity, and in cellulo function.