title: Oligonucleotides tethering Hoechst 33258 derivatives: effect of the conjugation site on duplex stabilization and fluorescence properties.
abstract: A series of DNA conjugates have been prepared in which two different derivatives of Hoechst 33258 have been tethered to a sequence containing a 5'-GAATTC-3' target site. The two derivatives differ only in the length of the tether between the DNA and the Hoechst fluorophore. By using a DNA backbone labeling protocol, one in which the Hoechst dye is tethered to an internucleotide phosphoramidate residue, it was possible to easily vary the site of attachment with respect to the A-T rich binding 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 end of the target sequence and finally within the AATT sequence, more effective helix stabilization results. When tethered between the two A residues, or between the A and T residue, a delta Tm of at least +20 degrees C is observed. Upon hybridization and formation of the B-form DNA, binding by the tethered Hoechst dye results, and the bound dye becomes brightly fluorescent. Upon a simple titration of the single-stranded conjugate with the complementary target sequence the quantum yield enhancement for hybridization only appears to be 5-7-fold at best. These 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 the single-stranded conjugate to a level comparable with that of the free 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.
authors.affiliation: Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
authors.forename: K
authors.initials: K
authors.lastname: Wiederholt
authors.forename: S B
authors.initials: SB
authors.lastname: Rajur
authors.forename: L W
authors.initials: LW
authors.lastname: McLaughlin
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D006690:Bisbenzimidazole / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D006690:Bisbenzimidazole