of template ODN 3. When ODNs 1c and 2 were irradiated
at 366 nm for 6 h in the presence of template ODN 3, the
clean formation of ODN 4c was observed by the densito-
metric assay of PAGE (Figure 5, lane 3). Further irradiation
Figure 3. Autoradiogram of a denaturing 15% polyacrylamide gel
electrophoresis of photoreaction of 32P-5′-end-labeled ODN 2 (7
µM) and ODN 1c (7 µM) and template ODN 3 (9 µM) in a solution
of NaCl (1 M) and sodium acetate buffer (50 mM, pH 5.0). (Lane
1) Authentic 32P-labeled photoligated product. (Lane 2) Before
photoligation. (Lane 3) Irradiation at 366 nm for 6 h, 0 °C, 89%
yield. (Lane 4) Irradiation at 366 nm for 6 h without template ODN
3. (Lane 5) Irradiation at 366 nm for 6 h, 70 °C.
Figure 5. Autoradiogram of a denaturing 15% polyacrylamide gel
electrophoresis of 32P-5′-end-labeled ODN 2 (7 µM) and ODN 1c
(7 µM) and template ODN 3 (9 µM) in a solution of NaCl (1 M)
and sodium acetate buffer (50 mM, pH 5.0). (Lane 1) Authentic
32P-labeled photoligated product. (Lane 2) Before photoligation.
(Lane 3) Irradiation at 366 nm for 6 h, 0 °C, 83% yield. (Lane 4)
Irradiation of lane 3 at 366 nm for 1 h in water/CH3CN ) 1:1 at
70 °C, 97% yield. (Lane 5) Incubation of lane 3 for 1 h in water/
CH3CN ) 1:1 at 70 °C.
densitometric assay of PAGE (Figure 3, lane 3). The enzy-
matic digestion of isolated ODN 4c, obtained from HPLC
purification, showed the formation of dC, dG, and dT in a
ratio of 3:4:3 together with YCVU-dT photoadduct, which was
confirmed by MALDI-TOF-MS (calcd 846.89 for [M - H]-;
found 846.81). The structure of YCVU-dT photoadduct ob-
tained from HPLC purification was assigned as a cis-syn
[2 + 2] adduct on the basis of spectroscopic data, including
1H-1H COSY and NOESY (see Supporting Information).
In the case of photoirradiation at 366 nm in the presence of
ODN 3 at 70 °C, no photoligated product was observed (lane
5). This result clearly shows that the photoligation via ODN
1c proceeded in a template-directed manner.
To examine the role of carbazole sensitizer in reversible
DNA photoligation, photoirradiation at 366 nm of ligated
ODN 4c was performed and analyzed by 15% PAGE. When
isolated ODN 4c was irradiated at 366 nm for 6 h in the
absence of template ODN 3, we observed the appearance of
ODN 2 in 90% yield, as determined by PAGE, along with
the disappearance of ODN 4c (Figure 4, lane 3). This
of lane 3 at 366 nm at 70 °C resulted in a complete reversion
to original ODN 2 (lane 4). Thus, these results indicate that
carbazole-tethered CVU-containing ODN can be used for
repeated DNA photoligation by the light-controlled photo-
reaction without any side reaction, such as the formation of
pyrimidine dimer. To examine the environment of the
carbazole in a DNA duplex, UV melting profiles were
obtained. The Tm value (46.8 °C) of the duplex of ODN
4c with ODN 3 was higher than that of ODN 3 and
the photoligated product from CVU-containing ODN
(5′-d(CTTCGTCVUGCGTG)-3′) (44.9 °C). Further, we per-
formed fluorescence titration of ODN 4c by ODN 3 in order
to study the environment of the carbazole (see Supporting
Information).12 The fluorescence intensity of carbazole was
gradually increased by 2.7-fold compared to that of the
single-stranded ODN 4c. These results suggested that the
carbazole in the duplex ODN 4c with ODN 3 intercalated
between bases and kept away from the photoligated position,
meaning DNA photoligation at 366 nm can proceed ef-
fectively in the duplex without carbazole-sensitized photo-
splitting. An important feature of this reversible DNA
photoligation is the fact that photoligation and photosplitting
at 366 nm can be controlled by the difference between the
single-stranded and duplex structures.
Figure 4. Autoradiogram of a denaturing 15% polyacrylamide gel
electrophoresis of photoreaction of ODN 4c (5 µM). (Lane 1)
Authentic 32P-5′-end-labeled ODN 2. (Lane 2) Before photosplitting.
(Lane 3) Irradiation at 366 nm for 6 h in water at ambient
temperature, 90% yield. (Lane 4) Irradiation at 366 nm for 1 h in
water/CH3CN ) 1:1 at ambient temperature, 99% yield. (Lane 5)
Irradiation of lane 3 at 366 nm for 6 h with template ODN 3 at 0
°C, 69% yield.
In conclusion, we have demonstrated that carbazole-
tethered CVU-containing ODN can be ligated by irradiation
at 366 nm in the presence of template ODN, and that the
(10) (a) Shin, J.-S.; Pierce, N. A. Nano Lett. 2004, 4, 905-909. (b) Le,
J. D.; Pinto, Y.; Seeman, N. C.; Musier-Forsyth, K.; Taton, T. A.; Kiehl,
R. A. Nano Lett. 2004, 4, 2343-2347.
(11) (a) Scannell, M. P.; Fenick, D. J.; Yeh, S.-R.; Falvey, D. E. J. Am.
Chem. Soc. 1997, 119, 1971-1977. (b) Joseph, A.; Prakash, G.; Falvey,
D. E. J. Am. Chem. Soc. 2000, 122, 11219-11225.
(12) (a) Hartshorn R. M.; Barton, J. K. J. Am. Chem. Soc. 1992, 114,
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photosplit ODN 1c can be ligated effectively to regenerate
the parent ODN 4c by photoirradiation at 366 nm in the
presence of template ODN 3 (lane 5). To demonstrate the
feasibility of this efficient and reversible photoligation, we
examined photoligation of ODNs 1c and 2 in the presence
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