DNA-TETHERED HOECHST GROOVE-BINDING AGENTS - DUPLEX STABILIZATION ANDFLUORESCENCE CHARACTERISTICS

Fluorescent Hoechst 33258 analogues have been synthesized in which the terminal phenol moiety is employed as a site for the introduction of a linker to permit covalent attachment of the fluorophores to oligo(de oxynucleotides). Hybridization by the DNA-Hoechst conjugates to target sequences generates the DNA minor groove structure and triggers a bin ding event by the tethered Hoechst agent. The attendant fluorescence s ignal reports upon this hybridization event. Conjugation of the Hoechs t derivatives to the DNA sequences employs a cystamine linker tethered to an internucleotide phosphorus residue. This mode of conjugation ma ximizes the versatility of linker placement and minimizes the associat ed chemistry required to introduce the linker. Two related Hoechst der ivatives have been synthesized; both contain a bromoacetamido linker f or conjugation to the oligonucleotides. With each Hoechst derivative, two pairs of diastereomeric (R(p) and S-p) oligo(deoxynucleotide) conj ugates were prepared to provide the tethered Hoechst groove binders wi th two different orientations within the dA-dT rich minor groove. T-m measurements suggest that while all of the conjugates provide some inc reased duplex stability, the diastereomeric conjugates tentatively ass igned the R(p) configuration exhibit nearly 20 degrees C increases in T-m values for the double-stranded dodecameric complexes, while those tentatively assigned as the S-p diastereomers exhibit only moderate 4- 8 degrees C increases in T-m values. The fluorescence characteristics of the conjugates are more variable, with one complex exhibiting a 23- fold enhancement in quantum yield effects, very similar to that observ ed for a free untethered Hoechst 33258 fluorophore bound to duplex DNA .