The examination of a key series of chromophore analogues of sandramycin (1) is detailed employing surface plasmon resonance to establish binding constants within a single high affinity bis-intercalation binding site 5'-d(GCATGC)2, and to establish the preference for sandramycin binding to 5'-d(GCXXGC)2 where XX=AT, TA, GC, and CG. From the latter studies, sandramycin was found to exhibit a preference that follows the order: 5'-d(GCATGC)2 > 5'-d(GCGCGC)2, delta deltaG(o)= 0.4 kcal/mol > 5'-d(GCTAGC)2, delta deltaG(o) = 0.9 kcal/mol> or =5'-d(GCCGGC)2, delta delta G(o) = 1.0 kcal/mol although it binds with high affinity to all four deoxyoligonucleotides. The two highest affinity sequences constitute repeating 5'-PuPy motifs with each intercalation event occurring at a 5'-PyPu step. The most effective sequence constitutes the least stable duplex, contains the sterically most accessible minor groove central to the bis-intercalation site, and the ability to accept two gly-NH/T C2 carbonyl H-bonds identified in prior NMR studies. Similarly, the contribution of the individual structural features of the chromophore were assessed with the high affinity duplex sequence 5'-d(GCATGC)2. In addition to the modest affinity differences, one of the most distinguishing features of the high affinity versus lower affinity bis-intercalation or mono-intercalation directly observable by surface plasmon resonance was the temporal stability of the complexes characterized by the exceptionally slow off-rates.