The sequence-specific structural and dynamic properties of double-helical DNA play important roles in many biological processes involving DNA recognition. Using a combination of NMR spectroscopy, surface plasmon resonance, and UV thermal denaturation experiments, we have investigated how sequences not making direct contact with the drug modulate the interaction between the cytotoxic agent elinafide and its preferred bisintercalation sites on double-helical DNA. Our combined data are consistent with two superposed interactions, one process involving ligand binding to the DNA duplex with nanomolar dissociation constants and another process of ring intercalation characterized by faster dissociation rates and substantially higher dissociation constants in some cases. The sequence of the base pairs flanking the bisnaphthalimide binding tetranucleotides influence both events through indirect readout effects, but these effects appear to be particularly relevant for the second (intercalation) process. The most unfavorable sequences contain specifically oriented A-tracts that oppose DNA intercalation of the naphthalimide rings, as reflected by strikingly different thermal stability and thermodynamic binding profiles. The complexes of elinafide with these sequences are characterized by poor DNA-naphthalimide and DNA-DNA stacking interactions and by enhanced dynamics of the ligand's intercalated rings and of the base pairs forming the tetranucleotide binding site.