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(deoxynucleotides). Hybridization by the DNA−Hoechst conjugates to target sequences generates the DNA minor groove structure and triggers a binding event by the tethered Hoechst agent. The attendant fluorescence signal reports upon this hybridization event. Conjugation of the Hoechst derivatives to the DNA sequences employs a cystamine linker tethered to an internucleotide phosphorus residue. This mode of conjugation maximizes the versatility of linker placement and minimizes the associated chemistry required to introduce the linker. Two related Hoechst derivatives have been synthesized; both contain a bromoacetamido linker for conjugation to the oligonucleotides. With each Hoechst derivative, two pairs of diastereomeric (R p and S p) oligo(deoxynucleotide) conjugates were prepared to provide the tethered Hoechst groove binders with two different orientations within the dA-dT rich minor groove. T m measurements suggest that while all of the conjugates provide some increased duplex stability, the diastereomeric conjugates tentatively assigned the R p configuration exhibit nearly 20 °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 °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 observed for a free untethered Hoechst 33258 fluorophore bound to duplex DNA.
A non-nucleoside linker based upon the ligand 2,2'-bipyridine and ethylene glycol is prepared and placed into the backbone of a number of oligonucleo-tides. The bipyridine ligand is reacted with cis -dichloro bis(2,2'-bipyridyl) Ru(II) to generate the relatively substitutionally inert complex based upon the well-characterized tris -2,2'-bipyridyl Ru(II). The ruthenium-containing DNA complexes exhibited UV and fluorescence characteristics that are consistent with those previously observed for simple tris -2,2'-bipyridyl Ru(II) complexes. Oligonucleotides containing the ruthenium complex will form both DNA duplexes and triplexes with stabilities that are slightly better than those formed from simple tethered oligonucleotide probes in which the two hybridizing sequences are tethered by simple tri(ethylene glycol) or hexa(ethylene glycol) linkers.
A fluorescent Hoechst 33258 derivative has been prepared in which a hexa(ethylene glycol) linker is attached to the terminal phenol residue. Conjugation of this derivative to DNA sequences is accomplished by a reversed coupling protocol, one in which the 5'-terminal nucleoside residue of a fully protected DNA sequence is converted to a terminal phosphoramidite. In the presence of the Hoechst derivative and tetrazole the final coupling reaction is achieved to generate the conjugated nucleic acid. After deprotection and cleavage of the conjugate from the support, HPLC analysis indicates that the conjugation reaction proceeds with yields as high as 75%. The presence of the conjugated Hoechst derivative increases the stability of DNA duplexes typically by 10-16 degrees C. A variety of sequence variants indicate that the tether length is sufficient to reach beyond the terminus of the DNA duplex and bind to internal A-T rich target sequences as far away as four base pairs from the site of attachment. A four base pair binding site appears to be necessary for effective helix stabilization by the conjugate, but in some cases can include a G-C base pair, which is consistent with a previous X-ray diffraction study regarding the binding of Hoechst 33258 to duplex DNA. When A-T base pairs alternate with G-C base pairs, a small but discernible increase is T(m) is observed (3.6 degrees C), indicating that binding to this sequence still occurs, but not in the same manner as to A-T rich sequences. Upon formation of the conjugated duplex, an enhanced quantum yield for the fluorescence emission spectrum of the tethered Hoechst derivative is observed. When an A-T rich binding site is present, the enhanced quantum yield increases by at least 16- and in some cases to nearly 30-fold relative to the value obtained for the single-stranded DNA-Hoechst conjugate.
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.
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