M. Sato et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1305–1308
1307
observed total lack of the stabilizing effect of dG in GK-
304 would be attributed to these facts. The result also
indicates that the current anthraquinone-bearing TFO
would be a particularly useful TFO against a pyr-
imidine-gap containing polypurine sequence when such
gap position is located near the end of the sequence.
The current anthraquinone-bearing phosphoramidite
derivative unit might be more advantageous compared
to the recently reported analogous amino acid deriva-
tive13 because of the ease in preparation and straight-
forward handling in an automated DNA-synthesizer
using phosphoramidite chemistry. The results presented
here indicate that novel modified oligonucleotides con-
taining an anthraquinone moiety as a non-nucleosidic
component would expand the utility of TFO as an effi-
cient and feasible gene-regulating agent. Optimization
of the structural feature of the conjugate including the
length of the linker portion is now underway and will be
reported elsewhere.
Figure 3. UV spectra of anthraquinone moiety incorporated into GK-
302. The solid line represents GK-302 (67 mM) alone. The broken line
represent GK-302 mixed with the double-stranded DNA (ODN-
2+ODN-3, 67 mM each) at 10 ꢀC. The samples were measured in the
same buffer system as it is indicated in Figure 2.
Table 1. Melting temperature (Tm) of the triplexes and the duplex
Acknowledgements
a
a
TFO
Triplex Tm
Duplex Tm
The work was partially supported by Grand-in-Aid for
Scientific Research from the Ministry of Education,
Culture, Sports, Science, and Technology, Japan.
GK-302
GK-303
GK-304
ꢀ-11merb
40.7
18.7
18.5
27.2
71.0
71.1
70.9
—
a Tm values (ꢀC) were determined by computer fitting of the first
derivative of the absorbance with respect to 1/T.
b The sequence of ꢀ-11mer is 50-TCT TTT TTT CT-30 which is
complementary to the polypurine region of the target sequence.11
References and notes
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observed a characteristic red shift (518–522 nm) of the
anthraquinone-based absorbance upon mixing of the
TFO and the dsDNA as it is shown in Figure 3. These
results suggest that the observed enhancement of the
stability of the triplex containing GK-302 is due to the
intercalation of the anthraquinone moiety to the target
dsDNA, to some extent. Interestingly, enhanced hyper-
chromicity was also clearly observed for the triplex-
duplex transition of GK-302 compared to GK-303 and
-304, as is also shown in Figure 2. This suggests that in
the triplex form, stronger stacking effect exists in GK-
302 compared to other TFO, which would also con-
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triplex. Another interesting observation here is that the
Tm values of the triplex containing either GK-303 or-304
are almost the same and no stabilizing effect of the tri-
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in GK-303 facing to the T–A pyrimidine gap position of
the dsDNA was observed. The result is somewhat con-
trary to the previous findings that dG-residue in a TFO
facing to the T–A pyrimidine gap stabilizes the triplex
through the formation of a single hydrogen bond with
T-residue.2a,12 It should be pointed out that, however,
the reported stabilizing effect of dG was observed when
the T–A gap is located near the central region of the
target polypurine strand which is not the case in this
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0
.
study. In addition, the 5 -neighboring triplet of G TA in
GK-304 is C+ GC which is known to destabilize the
.
G TA triplet to a considerable extent.12a,12c The
.