Abstract: Triplex complexes contain a single-stranded probe bound to a double-stranded nucleic acid target, in which the probe includes a heteropolymeric nucleic acid or a heteropolymeric nucleic acid analog. All base triplets of the complex are members selected from the group consisting of A-T-A, T-A-T, U-A-T, T-A-U, A-U-A, U-A-U, G-C-G and C-G-C. A cation-facilitated assay includes detecting the presence of such triplex complexes to determine the degree of complementarity between the probe and target sequence. The assay preferably detects a change in fluorescent intensity of a label as a function of binding affinity between the probe and target. The label can be covalently tethered to the probe or to the target, or can be an intercalating fluorophore in the reaction medium.