Massive parallel analysis of DNA-Hoechst 33258 binding specificity with a generic oligodeoxyribonucleotide microchip

A generic oligodeoxyribonucleotide microchip was used to determine the sequ ence specificity of Hoechst 33258 binding to double-stranded DNA, The gener ic microchip contained 4096 oxctadeoxynucleotides in which all possible 4(6 ) = 4096 hexadeoxy-nucleotide sequences are flanked on both the 3'- and 5'- ends with equimolar mixtures of four bases. The microchip was manufactured by chemical immobilization of presynthesized 8mers within polyacrylamide ge l pads. A selected set of immobilized 8mers was converted to double-strande d form by hybridization with a mixture of fluorescently labeled complementa ry 8mers. Massive parallel measurements of melting curves were carried out for the majority of 2080 6mer duplexes, in both the absence and presence of the Hoechst dye. The sequence-specific affinity for Hoechst 33258 was calc ulated as the increase in melting temperature caused by ligand binding, The dye exhibited specificity for A:T but not G:C base pairs. The affinity is low for two A:T base pairs, increases significantly for three, and reaches a plateau for four A:T base pairs. The relative ligand affinity for all tri nucleotide and tetranucleotide sequences (A/T)(3) and (A/T)(4) was estimate d. The free energy of dye binding to several duplexes was calculated from t he equilibrium melting curves of the duplexes formed on the oligonucleotide microchips. This method can be used as a general approach for massive scre ening of the sequence specificity of DNA-binding compounds.