Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)] and d(CCGGAATTCCGG): Multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities

摘要: Equilibrium binding experiments using fluorescence and absorption techniques have been performed throughout a wide concentration range (1 nM to 30 microM) of the dye Hoechst 33258 several DNAs. The most stable complexes found with calf thymus DNA, poly[d(A-T)], d(CCGGAATTCCGG), d(CGCGAATTCGCG) all dissociation constants in (1-3) X 10(-9) M-1. Such on DNA occur frequency about 1 site per 100 base pairs, evidence is presented indicating spectrum sequence-dependent affinities extending into micromolar range. In addition these sequence-specific sites continuous-variation method Job reveals distinct stoichiometries dye-poly[d(A-T)] corresponding 1, 2, 3, 4, 6 dyes 5 A-T pairs even up 2 (and possibly more) backbone phosphate. Models are suggested account for stoichiometries. With poly[d(G-C)] 1-2 G-C Thermodynamic parameters formation tightest complex between poly[d(A-T)] or d-(CCGGAATTCCGG) determined. chicken erythrocyte exhibits an ionic strength dependence similar that expected singly-charged positive ion. This remains unchanged presence 25% ethanol, which decreases affinity by orders magnitude. addition, due its strong binding, easily displaces intercalators from DNA.