Fluorescence titrations have been carried out to determine the association constants (Ka) for binding of the dyes Hoechst 33258 and DAPI to the self-complementary decamer d(CTGAATTCAG) and nine duplex derivatives with exocyclic substituent changes in the six central base pairs. Many Ka values are in the range (2-5) x 10(8) (duplex M)-1 at 5.5 degrees C. Replacement of the leftmost adenine by 2-aminopurine in the sequence decreases Ka for Hoechst 33258 by a factor of 170. When the centermost adenine is replaced by 2-aminopurine, Ka for Hoechst 33258 and DAPI is too small to be evaluated. When the centermost adenine is replaced by purine, Ka for both dyes increases, but this very stable duplex-Hoechst 33258 complex is nonfluorescent. The measured affinities are compared to expectations derived from X-ray studies with dodecamer-dye complexes having an identical central binding sequence (Pjura et al., 1987; Teng et al., 1988; Larsen et al., 1989).
2-Aminopurine Adenine