A light-controlled reversible DNA photoligation via carbazole-tethered 5-carboxyvinyluracil

Article abstract of DOI:10.1021/ol7026784

We describe a light-controlled template-directed reversible DNA photoligation via carbazole-tethered 5-carboxyvinyluracil. Carbazole-tethered 5-carboxyvinyl-2'-deoxyuridine-containing oligodeoxynucleotide (ODN) can be ligated by irradiation at 366 nm in the presence of template ODN, and the ligated ODN can be split by irradiation at 366 nm in the absence of template ODN.

Full text of DOI:10.1021/ol7026784

ORGANIC
LETTERS
2008
Vol. 10, No. 3
397-400
A Light-Controlled Reversible DNA
Photoligation via Carbazole-Tethered
5-Carboxyvinyluracil
Kenzo Fujimoto,*,† Hideaki Yoshino,‡ Takehiro Ami,† Yoshinaga Yoshimura,† and
Isao Saito‡,§
School of Materials Science, Japan AdVanced Institute of Science and Technology,
1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan, and Department of Synthetic
Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto UniVersity,
Kyoto 606-8501, Japan
kenzo@jaist.ac.jp
Received November 5, 2007
ABSTRACT
We describe a light-controlled template-directed reversible DNA photoligation via carbazole-tethered 5-carboxyvinyluracil. Carbazole-tethered
5-carboxyvinyl-2 -deoxyuridine-containing oligodeoxynucleotide (ODN) can be ligated by irradiation at 366 nm in the presence of template
ODN, and the ligated ODN can be split by irradiation at 366 nm in the absence of template ODN.
′
Template-directed DNA ligation that proceeds under the
control of specific DNA templates is an important technique
for potential synthetic and biotechnological applications.1 In
recent years, template-directed DNA ligation has developed
as a useful tool with applications such as nucleic acid
detection,2 sequence-specific small-molecule synthesis,3 and
the construction of nanoarchitecture.4 In our previous work,
we reported on template-directed reversible photoligation
with 5-carboxyvinyl-2′deoxyuridine (CVU) as a form of
phototriggered DNA manipulation.5 In this manipulation, the
ligated oligodeoxynucleotide (ODN) can be split site selec-
tively to regenerate the parent ODN by photoirradiation at
302 nm. Via these methods, we succeeded in the synthesis
of complex DNA structures, such as branched DNA, end-
capped DNA, or padlocked plasmid DNA, at the desired
sites.6 However, photoirradiation at 302 nm also results in
fatal damage to normal DNA, due to the formation of
pyrimidine dimer.7 Thus, one problem associated with
† Japan Advanced Institute of Science and Technology.
‡ Kyoto University.
§ Current address: NEWCAT Institute, School of Engineering, Nihon
University, Koriyama 963-8642, Japan.
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1995, 117, 10151-10152. (c) Xu, Y.; Karalkar, N. B.; Kool, E. T. Nat.
Biotechnol. 2001, 19, 148-152. (d) Czlapinski, J. L.; Sheppard, T. L. J.
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10.1021/ol7026784 CCC: $40.75
© 2008 American Chemical Society
Published on Web 01/09/2008

template-directed reversible DNA photoligation is the fact
that the efficiency of repeated DNA photoligation declines
gradually due to DNA photodamage. The development of a
photoligation system, which can be repeatedly ligated and
split, would represent a useful technique for DNA nano-
technology such as DNA nanoarchitecture,8 DNA comput-
ing,9 and DNA-based memory.10 N-Methylcarbazole was
known to be singlet excited-state electron donors, and
featured the (6-4) pyrimidine-pyrimidone lesion repaired
by photosensitized reductive electron-transfer reactions.11
Herein, we have overcome this problem by using carbazole
sensitizer-tethered CVU-containing ODN. We demonstrated
that control of photoligation and photosplitting at 366 nm is
possible, whether with or without the ODN template.
We initially determined the ability of the photosensitized
splitting with several photosensitizers, such as benzophenone,
riboflavin, N,N,N′,N′-tetramethylbenzidine, and N-methyl-
carbazole. When the photoligated ODN was irradiated at 366
nm in the presence of N-methylcarbazole, the photoligated
ODN was efficiently photosplit (see Supporting Information).
To examine the effect of varying the methylene linker arm
lengths between CVU and carbazole on the efficiency of DNA
photoligation, we designed three 6-mer ODNs with carbazole
photosensitizer-tethered CVU (5′-d(XGCGTG)-3′; ODN 1a,
ODN 1b, ODN 1c) as shown in Scheme 1. The phosphor-
on a DNA synthesizer, using phosphoramidite of carbazole-
tethered CVU. These modified ODNs were characterized by
a nucleoside composition and MALDI-TOF-MS (see Sup-
porting Information). When ODN 1a, ODN 1b, or ODN 1c
was used in the DNA photoligation, we observed the appear-
ance of the expected ligated 12-mer ODN 4a, ODN 4b, and
ODN 4c in 19, 57, and 65% yields, respectively (Figure 1).
These results indicate that the chain length can have a
considerable effect: -(CH2)6- > -(CH2)4- > -(CH2)2-.
Figure 1. Autoradiogram of a denaturing 15% polyacrylamide gel
electrophoresis of photoreaction of 32P-5′-end-labeled ODN 2 (7
µM) and ODN 1a, ODN 1b, and ODN 1c (each 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) Authentic 32P-labeled ODN 2. (Lane 3) Irradiation
at 366 nm to ODN 1a for 2 h, 0 °C, 19% yield. (Lane 4) Irradiation
at 366 nm to ODN 1b for 2 h, 0 °C, 57% yield. (Lane 5) Irradiation
at 366 nm to ODN 1c for 2 h, 0 °C, 65% yield.
Figure 2 shows a schematic chart for the light-controlled
reversible DNA photoligation via carbazole-tethered modi-
Scheme 1. Template-directed Photoligation of ODNs with
ODN 1a, ODN 1b, and ODN 1c
amidite of carbazole-tethered CVU was synthesized from
5-iodo-2′-deoxyuridine. These modified ODNs were syn-
thesized according to standard phosphoramidite chemistry
(6) (a) Fujimoto, K.; Yoshimura, Y.; Ikemoto, T.; Nakazawa, A.; Hayashi,
M.; Saito, I. Chem. Commun. 2005, 3177-3179. (b) Ogasawara, S.;
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Figure 2. Schematic representation of the light-controlled revers-
ible DNA photoligation.
(8) (a) Shih, W. M.; Quispe, J. D.; Joyce, G. F. Nature 2004, 427, 618-
621. (b) Goodman, R. P.; Schaap, I. A. T.; Tardin, C. F.; Erben, C. M.;
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fied ODNs. When ODN 1c and 32P-5′-end-labeled 6-mer
(ODN 2) were irradiated at 366 nm for 6 h in the presence
of template 12-mer (ODN 3), the expected ligated 12-mer
ODN 4c was produced at a 89% yield as determined by the
398
Org. Lett., Vol. 10, No. 3, 2008

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
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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
Org. Lett., Vol. 10, No. 3, 2008
399

ligated ODN can be split by irradiation at 366 nm in the
absence of template ODN. This light-controlled template-
directed reversible DNA photoligation may provide a unique
methodology for DNA engineering, DNA nanotechnology,
and photochemical DNA manipulation.
Supporting Information Available: Synthetic procedures
for carbazole-tethered modified ODNs, experimental pro-
cedures for the photoligation and photosplitting, spectro-
scopic structural determination of YCVU-dT photoadduct,
detailed experimental data of fluorescence titration. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. This work was supported by a Grant-
in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science, and Technology, Japan.
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