Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities.
Equilibrium binding experiments using fluorescence and absorption techniques have been performed throughout a wide concentration range (1 nM to 30 microM) of the dye Hoechst 33258 and several DNAs. The most stable complexes found with calf thymus DNA, poly[d(A-T)], d(CCGGAATTCCGG), and d(CGCGAATTCGCG) all have dissociation constants in the range (1-3) X 10(-9) M-1. Such complexes on calf thymus DNA occur with a frequency of about 1 binding site per 100 base pairs, and evidence is presented indicating a spectrum of sequence-dependent affinities with dissociation constants extending into the micromolar range. In addition to these sequence-specific binding sites on the DNA, the continuous-variation method of Job reveals distinct stoichiometries of dye-poly[d(A-T)] complexes corresponding to 1, 2, 3, 4, and 6 dyes per 5 A-T base pairs and even up to 1 and 2 (and possibly more) dyes per backbone phosphate. Models are suggested to account for these stoichiometries. With poly[d(G-C)] the stoichiometries are 1-2 dyes per 5 G-C pairs in addition to 1 and 2 dyes per backbone phosphate. Thermodynamic parameters for formation of the tightest binding complex between Hoechst 33258 and poly[d(A-T)] or d-(CCGGAATTCCGG) are determined. Hoechst 33258 binding to calf thymus DNA, chicken erythrocyte DNA, and poly[d(A-T)] exhibits an ionic strength dependence similar to that expected for a singly-charged positive ion. This ionic strength dependence remains unchanged in the presence of 25% ethanol, which decreases the affinity by 2 orders of magnitude. In addition, due to its strong binding, Hoechst 33258 easily displaces several intercalators from DNA.
Laboratory of Biochemistry, Faculty of Sciences, Gent State University, Belgium.
F G
FG
Loontiens
P
P
Regenfuss
A
A
Zechel
L
L
Dumortier
R M
RM
Clegg
D000042:Absorption; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001667:Binding, Competitive; D006690:Bisbenzimidazole / Q000378:metabolism*; D002417:Cattle; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011067:Poly dA-dT / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013950:Thymus Gland / Q000737:chemistry
D011067:Poly dA-dT; D004247:DNA; D006690:Bisbenzimidazole