Circular dichroism and thermal melting differentiation of Hoechst 33258 binding to the curved (A(4)T(4)) and straight (T(4)A(4)) DNA sequences

The ability of the B-DNA minor groove ligand Hoechst 33258 to discriminate between prototype curved and straight duplex DNA sequences was investigated by circular dichroism (CD) titrations at the wavelengths of absorbance of the ligand. The sequences were studied either within the framework of the ligated decamers (CA(4)T(4)G)(n) and (CT(4)A(4)G)(n), or within that of the single dodecamers GCA(4)T(4)GC and GCT(4)A(4)GC, to confirm and extend our earlier results based on fluorescence titrations of ligated decamers. A unique, strong binding site is invariantly present in both sequence units. The binding affinity of the drug for the site in the curved A(4)T(4) sequence was found 3- to 4-fold higher compared to the straight sequence. All these features hold true irrespective of the sequence framework, thus confirming that they reflect specific properties of the binding to the two sequences. Ligand binding increases the thermal stability of straight and curved duplex dodecamers to the same extent, thus maintaining the melting temperature differential between the two sequences. However, the different melting patterns and the difference between total ligand]:site] ratios needed for site saturation in the two duplexes are in agreement with the difference between binding constants derived from CD measurements.