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-OHassociation 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 x 10(9 )M(-1) and bis-m-OH 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 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 x 10(8) M(-1) x s(-1) to 2.9 x 10(8) M(-1) x 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 x 10(7) M(-1) x s(-1) and 5.2 x 10(8) M(-1) x s(-1)) depends on the DNA.