A. T. M. Zafrul Azam et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5747–5750
Table 1. Melting temperature (Tm) of the triplexes and the duplex
5749
Next, the UV-absorbance at 535nm in Figure 3a was
plotted against temperature and the result is shown in
Figure 3b. As is clearly seen from Figure 3b, the plot
shows prominent increment of the absorbance between
50 and 60°C. The essentially same results were obtained
from the corresponding experiment for other modified
TFOs. The obtained plots reflect the association and dis-
sociation of the intercalator moiety from the target
dsDNA, presumably. The results strongly suggest that
the intercalator moiety in the modified TFOs works as
a molecular anchor to fasten the TFO to the target.
a
a
b
TFO
Triplex Tm
Duplex Tm
DTm
GK-300
GK-329
GK-330
GK-331
GK-332
38.6
49.4
49.0
52.1
51.2
71.1
71.3
71.1
71.1
70.9
—
10.8
10.4
13.5
12.6
a Tm values (°C) were determined by computer fitting of the first
derivative of the absorbance with respect to 1/T.
b DTm indicates the deviation from the Tm value of the corresponding
triplex composed of GK-300 and the duplex.
In conclusion, we have successfully demonstrated the
preparation and the remarkable triplex-stabilizing abil-
ity of novel a–b chimeric oligo-DNA bearing an interca-
lator-conjugated nucleobase located at the linker region
as a unique alternate-stranded triplex forming oligonu-
cleotide. The triplex-stabilizing ability of the modified
chimera DNA arises from the intercalation of the inter-
calative moiety.
modified TFO, GK-331 having the adjacent modified
bases exhibited the highest stabilizing effect, although
the difference in the Tm between GK-331 and 332 was
only about 1°C. Presumably, the observed Tm increment
effect of the modified TFO is due to the interaction of
the anthraquinone moiety and the double-stranded por-
tion of the target dsDNA.
To confirm such interaction, we carried out a set of
experiments monitoring the change in UV-absorption
of the anthraquinone moiety. Thus, the complex formed
between GK-332 and the dsDNA was heated at elevated
temperature and the UV-absorbance at the range of
400–700nm was monitored. As shown in Figure 3a,
The ease in preparation and the substantial triplex-stabi-
lizing effect of the current modified chimera DNA would
make it more advantageous compared to the previously
reported modified chimera bearing a dinucleoside phos-
photriester unit conjugated with the same intercalative
moiety having hexamethylene tether group at its inter-
nucleotide linkage in a stereospecific manner.8 Also,
the results presented here indicate that the C-4 position
of the pyrimidine base in the linker portion of the chi-
mera is a very suitable position to incorporate a certain
molecule capable of interacting with dsDNA, such as
DNA cleaving molecule.
the anthraquinone-based absorbance exhibited
a
marked shift to the shorter wave length (blue shift)
according to the elevation of the temperature. At the
same time, the absorption maximum was also increased
(hyperchromic effect). A combination of these character-
istic behaviors in UV-spectroscopy strongly suggests
that the anthraquinone moiety actually intercalates to
the target dsDNA when the modified TFO binds it to
form the triplex.
Optimization of the structural feature of the conjugate
including the length of the linker portion is now under
way and will be reported elsewhere.
Acknowledgements
The work was partially supported by Grant-in-Aid for
Scientific Research from the Ministry of Education, Cul-
ture, Sports, Science, and Technology, Japan. K.S. also
thanks the Ministry for supporting A.Z.A. as a scholar
of Monbukagakusho Scholarship for Foreign Graduate
Students.
References and notes
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Figure 3. Temperature dependent UV-change of anthraquinone moi-
ety in GK-332. The experiment was carried out in the same condition
as the UV-melting experiment, although the concentration of the
triplex was 18.6lM.
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