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The binding of 9-aminoacridine and one bis-acridine compound to double helical poly(dA).poly-(dT) and poly[d(A-T)]2 and triple helical poly(dA).[poly(dT)]2 has been investigated using linear dichroism (LD) and circular dichroism (CD). A close examination of the negative reduced LD and the induced CD for the first pi-->pi* transition absorption region leads us to conclude that the acridine moiety of the 9-aminoacridine and bis-acridine molecule intercalates with both duplex and triplex DNA. Binding geometries of the acridine moieties in the examined polynucleotides are similar to those found for the ligands with DNA (Hansen et al. (1984) J. Chem. Soc., Chem. Commun., 509-511). It is also found that both 9-aminoacridine and bis-acridine effectively enhance the thermal stability of the triplex DNA. The corresponding spectra for the complexes of the minor groove binders DAPI and Hoechst with poly-(dA).[poly(dT)]2 were studied for comparison. They both show a positive LD and a mixing ratio dependent positive CD in the ligand absorption region, similar to those of their duplex complexes. This indicates that these ligands bind in the grooves of the triplex, probably to the one corresponding to the minor groove of the template duplex.