Binding of Hoechst 33258 and 4',6'-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents.

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).