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.
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 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.
Breusegem
S Y
SY
Laboratory for Biochemistry Department of Biochemistry Physiology and Microbiology Faculty of Sciences, Ghent University, Ledeganckstraat, Gent, B-9000, Belgium.
Sadat-Ebrahimi
S E
SE
Douglas
K T
KT
Clegg
R M
RM
Loontiens
F G
FG
D020856:AT Rich Sequence / Q000235:genetics; D000327:Adsorption; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004058:Diffusion; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D006207:Half-Life; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010636:Phenols / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D017186:Titrimetry
D005456:Fluorescent Dyes; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D010636:Phenols; D004247:DNA