HOECHST-33258 TETHERED BY A HEXA(ETHYLENE GLYCOL) LINKER TO THE 5'-TERMINI OF OLIGODEOXYNUCLEOTIDE 15-MERS - DUPLEX STABILIZATION AND FLUORESCENCE PROPERTIES
Sb. Rajur et al., HOECHST-33258 TETHERED BY A HEXA(ETHYLENE GLYCOL) LINKER TO THE 5'-TERMINI OF OLIGODEOXYNUCLEOTIDE 15-MERS - DUPLEX STABILIZATION AND FLUORESCENCE PROPERTIES, Journal of organic chemistry, 62(3), 1997, pp. 523-529
A fluorescent Hoechst 33258 derivative has been prepared in which a he
xa(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 re
sidue of a fully protected DNA sequence is converted to a terminal pho
sphoramidite. In the presence of the Hoechst derivative and tetrazole
the final coupling reaction is achieved to generate the conjugated nuc
leic acid. After deprotection and cleavage of the conjugate from the s
upport, HPLC analysis indicates that the conjugation reaction proceeds
with yields as high as 75%. The presence of the conjugated Hoechst de
rivative increases the stability of DNA duplexes typically by 10-16 de
grees C. A variety of sequence variants indicate that the tether lengt
h is sufficient to reach beyond the terminus of the DNA duplex and bin
d 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 3325
8 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), ind
icating that binding to this sequence still occurs, but not in the sam
e manner as to A-T rich sequences. Upon formation of the conjugated du
plex, an enhanced quantum yield for the fluorescence emission spectrum
of the tethered Hoechst derivative is observed. When an A-T rich bind
ing 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.