A series of pyrrole, imidazole-substituted bis-benzimidazole conjugates, Py-Py-im-gamma-biBenz, PyPy-gamma-biBenz, Py-Im-gamma-biBenz, and Im-Py-gamma-biBenz (1-4), were prepared in an attempt to target dsDNA sequences possessing both A/T and G/C bps. The dsDNA interactions and sequence specificity of the conjugates have been characterized via spectrofluorometric titrations and thermal melting studies. All conjugates form 1:1 complexes with dsDNA at subnanomolar concentrations. The Im moiety selectively recognizes a G/C bp embedded in the A/T-rich binding site. This represents the first clear example of sequence selective recognition in a 1:1 motif.(1) The equilibrium association constant (K-1) for complexation of a specific nine-bp dsDNA site, 5'-gcggTATGAAATTcgacg-3', by conjugate 1 is similar to2.6 x 10(9) M-1. Displacement of the G/C position or G/C-->A/T substitution within the nine-bp site decreases the K, by similar to8-fold, whereas two continuous G/C bps decrease the K, by similar to50-fold magnitude. The K, values for seven-bp dsDNA, 5'-gcggtaTGAAATTcgacg-3' and 5'-gcggtaCAAAATTcgacg-3', binding sites by conjugates Py-Im-gamma-biBenz (3) and lm-Py-gamma-biBenz (4) are similar to2.3 x 10(9) and similar to1.2 x 10(9) M-1, respectively. However, the conjugates with no Im moiety, Py-Py-gamma-biBenz (2) and Py-Py-Py-gamma-biBenz (5 and 6), are specific for seven-to nine-bp A/T-rich sites and single A/T-->G/C bp substitution within the binding site decreases the K, values by 1-2 orders of magnitude.