Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics

Abstract

The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH < m-OCH3, p-OH < m-OH < m-OH, p-OCH3 < bis-m-OH. The association constants Ka are three to 11 times larger for the AATT site than for poly[d(A-T)]. The AATT site binds m-OH Hoechst with Ka = 3.8 × 109 M−1 and bis-m-OH Hoechst with Ka = 1.9 × 1010 M−1, which are seven and 37 times higher than p-OH Hoechst (Hoechst 33258), respectively. The high Ka values determined at equilibrium agree with the kinetically defined association constants Kkin = kon/koff. The association-rate parameters kon were obtained by stopped-flow kinetics and the dissociation-rate parameters koff by dissociation kinetics using poly[d(A-5BrU)]. For binding to the AATT site, kon values are similar and nearly diffusion-controlled (2.0 × 108 M−1 s−1 to 2.9 × 108 M−1 s−1), while koff values (0.42 s−1 to 0.012 s−1) depend on the phenyl substitution and determine the affinity. At the AATT site, the longest-living complex is formed when the dye carries a bis-m-OH phenyl group that probably integrates in a hydrogen-bonding network of water molecules. With poly(dA)·poly(dT), poly[d(A-T)] and poly[d(A-5BrU)], kon (between 6.1 × 107 M−1 s−1 and 5.2 × 108 M−1 s−1) depends on the DNA.