Since the discovery of the bioactive natural-product antitumor–antibiotics echinomycin and triostin A, bisintercalating molecules have attracted considerable attention in the biomedical field. We are interested in designing metallobisintercalators for applications in cancer chemotherapy. Previously, a bisintercalative DNA-binding mode has been demonstrated for cytotoxic PT-BIS(ACRAMTU) (1) ([Pt(en)(ACRAMTU)₂](NO₃)₄; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1, 3-dimethylthiourea, en = ethylene-diamine), a bifunctional platinum-acridine conjugate, using various biophysical methods and NMR spectroscopy. Structure–activity relationship (SAR) studies of newly synthesized cis and trans derivatives of PT-BIS(ACRAMTU) reveal that the trans derivative 2, which contains threading groups and carries a high cationic charge, shows ~45-fold increased cytotoxicity compared to the prototype in HL-60 leukemia cells. CD spectroscopic studies of a small library of conjugates indicate that a relationship exists between the cell-kill potential of these agents and their ability to induce an unusual DNA conformational switch: in alternating GC sequences, conjugate 2 induces a right-handed non-B-form conformation containing Hoogsteen base pairing. Trapping or stabilizing of this conformation, which may be present as a transient form during transcription and replication, may block the progression of polymerase, a potential cytotoxic event. The biological consequences are discussed in detail.