DNA-Tethered Hoechst Groove-Binding Agents: Duplex Stabilization and Fluorescence 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(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
(Rp and Sp)
oligo(deoxynucleotide) conjugates were prepared
to provide the tethered Hoechst groove binders with two different
orientations within the dA-dT rich minor groove.
Tm measurements suggest that while all of
the conjugates provide some increased duplex stability, the
diastereomeric
conjugates tentatively assigned the Rp
configuration exhibit nearly 20 °C increases in
Tm values for the double-stranded dodecameric complexes, while those tentatively assigned as the
Sp diastereomers exhibit only
moderate
4−8 °C increases in Tm 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.