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

The large increase in fluorescence upon binding of five para- and metapheny l 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-f low 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 const ants 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 x 10(9) M-1 and bis-m-O H Hoechst with K-a = 1.9 x 10(10) M-1, which are seven and 37 times higher than p-OH Hoechst (Hoechst 33258), respectively. The high K-a values determ ined at equilibrium agree with the kinetically defined association constant s 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 di ssociation kinetics using poly[d(A-5BrU)]. For binding to the AATT site, k( on) values are similar and nearly diffusion-controlled (2.0 x 10(8) M-1 s(- 1) to 2.9 x 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 AAT T site, the longest-living complex is formed when the dye carries a bis-m-O H phenyl group that probably integrates in a hydrogen-bonding network of wa ter molecules. With poly(dA).poly(dT), poly[d(A-T)] and poly[d(A-5BrU)], k( on) (between 6.1 x 10(7) M and 5.2 x 10(8) M-1 s(-1)) depends on the DNA. ( C) 2001 Academic Press.