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 T 4 -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 -OCH 3 , p -OH m -OH m -OH, p -OCH 3 m -OH. The association constants K a are three to 11 times larger for the AATT site than for poly[d(A-T)]. The AATT site binds m -OH Hoechst with K a = 3.8 × 10 9 M −1 and bis- m -OH Hoechst with K a = 1.9 × 10 10 M −1 , which are seven and 37 times higher than p -OH Hoechst (Hoechst 33258), respectively. The high K a values determined at equilibrium agree with the kinetically defined association constants K kin = k on / k off . The association-rate parameters k on were obtained by stopped-flow kinetics and the dissociation-rate parameters k off by dissociation kinetics using poly[d(A-5BrU)]. For binding to the AATT site, k on values are similar and nearly diffusion-controlled (2.0 × 10 8 M −1 s −1 to 2.9 × 10 8 M −1 s −1 ), while k off 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)], k on (between 6.1 × 10 7 M −1 s −1 and 5.2 × 10 8 M −1 s −1 ) depends on the DNA.