OLIGONUCLEOTIDES TETHERING HOECHST-33258 DERIVATIVES - EFFECT OF THE CONJUGATION SITE ON DUPLEX STABILIZATION AND FLUORESCENCE PROPERTIES

A series of DNA conjugates have been prepared in which two different d erivatives of Hoechst 33258 have been tethered to a sequence containin g a 5'-GAATTC-3' target site. The two derivatives differ only in the l ength of the tether between the DNA and the Hoechst fluorophore. By us ing a DNA backbone labeling protocol, one in which the Hoechst dye is tethered to an internucleotide phosphoramidate residue, it was possibl e to easily vary the site of attachment with respect to the A-T rich b inding site. When tethered outside the GAATTC sequence, little if any helix stabilization results upon hybridization of the conjugate to its complementary sequence. As the site of conjugation is moved to one en d of the target sequence and finally within the AATT sequence, more ef fective helix stabilization results. When tethered between the two A r esidues, or between the A and T residue, a Delta T-m of at least +20 d egrees C is observed. Upon hybridization and formation of the B-form D NA, binding by the tethered Hoechst dye results, and the bound dye bec omes brightly fluorescent. Upon a simple titration of the single-stran ded conjugate with the complementary target sequence the quantum yield enhancement for hybridization only appears to be 5-7-fold at best. Th ese fluorescence effects, generally less dramatic than those observed with other sequences, result from an increase in quantum yield for the single-stranded conjugate relative to the free Hoechst 33258. Heating the single-stranded conjugate reduces the inherent fluorescence of th e single-stranded conjugate to a level comparable with that of the fre e Hoechst dye. In experiments monitoring absorbance vs temperature, a cooperative transition is observed for the single-stranded conjugate. Both the high quantum yield observed for the single-stranded conjugate and the observed thermally induced transition suggest that the single -stranded conjugate can dimerize (at the GAATTC site), mediated by the groove-binding fluorophore.