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Title

Site‑specific transition of cytosine to uracil
via reversible DNA photoligation

Author(s)Fujimoto, Kenzo; Matsuda, Shigeo; Yoshimura,
Yoshinaga; Matsumura, Takashi; Hayashi, Masayuki;
Saito, Isao

Citation

Chemical Communications, 2006(30): 3223‑3225

Issue Date

2006

Type

Journal Article

Text version

author

URL

http://hdl.handle.net/10119/7905

RightsCopyright (C) 2006 Royal Society of Chemistry.
Kenzo Fujimoto, Shigeo Matsuda, Yoshinaga
Yoshimura, Takashi Matsumura, Masayuki Hayashi
and Isao Saito, Chemical Communications,
2006(30), 2006, 3223‑3225.
http://dx.doi.org/10.1039/b605289d ‑ Reproducedby permission of The Royal Society of Chemistry

Description

Japan Advanced Institute of Science and Technology

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Site-specific Transition of Cytosine to Uracil via Reversible DNA
Photoligation
Kenzo Fujimoto,*ab Shigeo Matsuda,c Yoshinaga Yoshimura,b Takashi Matsumura,a Masayuki Hayashid
and Isao Saitoe
Receipt/Acceptance Data [DO NOT ALTER/DELETE THIS TEXT]
Publication data [DO NOT ALTER/DELETE THIS TEXT]DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT]

We report that deamination coupled with 5-carboxyvinyldeoxyuridine-mediated photobranching causes the heat-induced
transition of cytosine to uracil with high efficiency without any
side reaction.
The development of a technique for artificial site-specific mutation
will be a useful genetic tool for preparing non-native proteins. The
mutation of nucleic acid bases is also caused by intrinsic factors
such as sunlight and ultraviolet light.1 In living dells, transition of C
is also caused by the formation of pyrimidine dimer in the 5’-CT-3’,
5’-TC-3’ and 5’-CC-3’ sequence by UV irradiation coupled withenzymatic repair.2,3,4 The deamination of cytosine in cyclobutane
pyrimidine dimers was assigned as a key step in these transitions
based on the reduced half-life of deamination to a matter of hours
when the 5,6 bond is saturated compared to the 30,000 years’ halflife for the deamination of normal monomeric cytosine. While
many kinetic and biological studies on such deamination havealready been reported,5–13 a method for C to U transition based on
this deamination process with site-specificity has not been reported.
We have recently reported a template-directed DNA photoligation
mediated by 5-vinyldeoxyuridine (VU) via [2+2] cycloaddition
between the VU base and terminal C.14 In fact, the rapid
deamination of the photoligated dimer in an enzymatic digestion
process has been also observed in this study.14 We have also
reportedthephotochemical
synthesisofbranched
oligodeoxynucleotides (ODNs) via 5-carboxyvinyldeoxyuridine(CVU).14 We therefore report here the artificial site-specifictransition from cytosine to uracil mediated by reversible DNA
photoligation. The efficient, selective transition of cytosine to uracil
was achieved by photochemical branched ODN formation at thetarget cytosine and the successive photosplitting of the branched
ODN after heat treatment.
CV
U-containing ODN was synthesized according to standard
phosphoramidite chemistry on a DNA synthesizer using
phosphoroamidite of methyl ester of CVU coupled with the postmodification procedure after ODN synthesis (Scheme 1).15 Figure 1
shows a schematic chart for the method for site-selective transition
from cytosine to uracil and the detection system of uracil as acleavage band in PAGE analysis. The photoirradiation of cytosinecontaining ODN at 366 nm while addressing the CVU-containing
hairpin template ODN introduced the branch structure at the target
cytosine via [2+2] cycloaddition between the CVU base and thetarget cytosine. The deamination of the target cytosine waspromoted site-specifically with heat treatment to produce a uracil
analogue in ligated ODN. After heat treatment, uracil-containing
ODN was produced by photosplitting at 302-nm irradiation. The
uracil base produced in ODN was removed with uracil DNA
glycosylase (UDG), and the resulting abasic site was converted into
† Electronic Supplementary Information (ESI) available: Experimental
details. See http://www.rsc.org/suppdata/xx/b0/b000000x/* kenzo@jaist.ac.jp

This journal © Royal Society of Chemistry

Scheme 1 ODNs used in this study.

Fig. 1 Outline of the method for site-specific transition and thedetection of uracil.
strand breaks with piperidine treatment, which was detected by
PAGE analysis (Fig. 1).16 When 32P-5'-end-labeled 11mer 5’[JOURNAL], 200X, 00, 0000 | 1

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Communicationd(TGTGCAAAAAA)-3’
(ODN
1)
and
21mer
5’d(CVUGCGTGTTTTCACGCAGCACA)-3’ (ODN 2) were
irradiated at 366 nm for 2 h (Fig. 1), the expected ligated 32mer
ODN 3 was produced at a 98% yield as determined by thedensitometric assay of PAGE (Fig. 2, lane 2). After heating at
90 °C for 2 h (Fig. 2, lane 3), quantitative photosplitting wasachieved by 302-nm irradiation for 1 h (Fig. 2, lane 4). A cleavage
band was observed by piperidine treatment after enzymatic baseexcision using UDG after photosplitting (Fig. 2, lane 5). The
mobility of this band was equal to the band that was produced after
strand cleavage at the abasic sites by the UDG treatment ofauthentic deoxyuridine-containing ODN 4 (Fig. 2, lane 8). Thiscleaved band was not observed when the heating of photoligatedODN 3 at 90 °C was not conducted (Fig. 2, lane 6). These results
clearly indicate that the cleaved band seen in lane 5 was caused by
the transformation from cytosine to uracil at the target site via thedeamination of the C<>CVU photoadduct by heating at 90 °C andphotosplitting at 302 nm. This method for the transition of cytosine
to uracil proceeded effectively at every step. Uracil formation was
also directly confirmed by the HPLC analysis of enzymatic
digestion of isolated ligated 17mer after heat treatment.

Fig. 2Autoradiogram of denaturing polyacrylamide gelelectrophoresis of site-selective cytosine to the uracil mutation ofCV
U-containing ODN 2 (7 µM) and 32P-5'-end-labeled ODN 1 (1µM) in a sodium cacodylate buffer (50 mM, pH 7.0) containing
NaCl (100 mM). Lane 1, before reaction; lane 2, ODN 1 + ODN 2,
irradiation at 366 nm, 3 h on ice; lane 3, incubation at 90 °C for 2 hof lane 2; lane 4, irradiation at 302 nm, 1 h at 40 °C of lane 3; lane
5, UDG treatment at 37 °C for 1 h followed by incubation withpiperidine at 90 °C for 20 min of lane 4; lane 6, irradiation at 302
nm, 1 h of lane 2; lane 7, UDG treatment followed by piperidine
treatment of lane 6 under the same conditions as lane 5; lane 8,
treatment of ODN 4 with UDG and piperidine under the same
conditions as lane 5.
The temperature dependency of this deamination process of
C<>CVU photoadduct-containing ODN 3 was examined in duplex
in order to understand the deamination kinetics. The deamination
reaction was conducted at 37 °C and 90 °C, and the efficiency wasestimated by quantifying the intensity of the cleaved band after
UDG treatment as described above. The rate constant for
deamination was 2.75 × 10–4 at 90 °C, pH 7.0, which was twoorders of magnitude greater than that of 2.66 × 10–6 at 37 °C, pH
7.0 for deamination.17 This acceleration in the rate constant for
deamination is likely attributed not only to a gain in its activationenergy for deamination but also to the increased accessibility of thecytosine analogue in denatured DNA to protons and hydroxyl ions.
The rate constant for deamination at 37 °C was similar to that at 1.8
× 10–6 observed in the CCCC target sequence containing
2 | [JOURNAL], 200X, 00, 0000

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M13mp2C141 ds DNA exposed to UV at 37 °C, pH 7.4.7 Thissuggests the similarity of solvent exposure originating in local
structural and conformational changes between C<>VC dimercontaining ODN in duplex and C<>C dimer-containing ODN in
duplex. The effect of pH for deamination was also examined at
37 °C. However no difference was observed in its rate constant for
deamination at pH 5.0, pH 7.0 and pH 9.0. This indicates that the
protonation of N3 of the cytosine analogue and attack by water orhydroxyl at C4 of the cytosine analogue in this addition elimination
pathway of deamination might also play an important role in the
kinetics of deamination. These results indicate that the cytosine
residue in the C<>CVU photoadduct was easily transformed touracil residue at higher temperatures.7

Fig. 3 Autoradiogram of a denaturing polyacrylamide gelelectrophoresis of site-specific cytosine to the uracil mutation ofCV
U-containing ODN 6 (7 µM) and 32P-5'-end-labeled ODN 5 (1µM) in a sodium cacodylate buffer (50 mM, pH 7.0) containing
NaCl (100 mM). Lane 1, before reaction; lane 2, ODN 5 + ODN 6,
irradiation at 366 nm, 3 h on ice; lane 3, incubation at 90 °C for 2 hof lane 2; lane 4, irradiation at 302 nm, 1 h at 40 °C of lane 3; lane
5, UDG treatment at 37 °C for 1 h followed by incubation withpiperidine at 90 °C for 20 min of lane 4; lane 6, irradiation at 302
nm, 1 h of lane 2; lane 7, UDG treatment followed by piperidine
treatment of lane 6 under the same conditions as lane 5; lane 8,
treatment of ODN 8 with UDG and piperidine under the same
conditions as lane 5; lane 9, incubation with piperidine at 90 °C for
20 min of lane 4.
We further demonstrate that deamination coupled with CVUmediated photobranching can be carried out when ODN containing
two consecutive cytosines is used as a target sequence. When 32P5'-end-labeled 20mer 5’-d(TGGCTACGAGCCAACAACAA)
(ODN
5)
and
27mer
5’d(CVUGCGTGTTTTCACGCAGCTCGTA GCCA) (ODN 6) were
irradiated at 366 nm for 3 h, the expected ligated 47mer ODN 7
was produced in 80% yield as determined by densitometric assay of
PAGE (Fig. 3, lane 2). After heating at 90 °C for 2 h (Fig. 3, lane 3),
quantitative photosplitting was achieved by 302-nm irradiation for
1 h (Fig. 3, lane 4). A cleavage band was observed by piperidine
treatment after enzymatic base excision using UDG after
photosplitting (Fig. 3, lane 5). The mobility of this band was equal
to the band that was produced after the strand cleavage at the abasic
sites produced by UDG treatment of authentic deoxyuridinecontaining ODN 8 (Fig. 3, lane 8). This cleaved band was notobserved when heating of photoligated ODN 7 at 90 °C was not
carried out (Fig. 3, lane 6) or when piperidine treatment was carriedThis journal © Royal Society of Chemistry

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out without UDG treatment after photosplitting (Fig. 3, lane 9).
These results clearly indicated that the cleaved band seen in lane 5
was caused by the transformation from cytosine to uracil at thetarget site via deamination of C<>CVU photoadduct by heating at
90 °C and photoplitting at 302 nm. It was also found that an
efficient transformation from cytosine to uracil occurred at thetarget cytosine at eleventh position with site-selectivity. Thesedeaminations of C at eleventh position and C at twelfth position
were 88% and 12% yield, respectively.
The heat-induced deamination of the cytosine residue of acyclobutane dimer followed by splitting of the photolyase7,8,12 orphotoreactivation2,4,11 has thus far been known as a method forconverting cytosine to uracil. However, these methods are not sitespecific. Our deamination method using reversible DNA
photoligation has high sequence selectivity and efficiency at thetarget cytosine compared with other methods for converting
cytosine to uracil using enzymatic or chemical procedures.5,7,11
In summary, we have demonstrated that deamination coupledwith CVU-mediated photobranching causes the heat-induced
transition of cytosine to uracil with high efficiency without any side
reaction. The present site-specific C to U transition can be widelyused for the site-specific mutation of DNA. This method may alsofind application in mutation not only for DNA but also formessenger RNA.

Communication
13 M. M. Ali, M. R. Alam, T. Kawasaki, S. Nakayama, F. Nagatsugi
and S. Sasaki, J. Am. Chem. Soc., 2004, 126. 8864.
14 K. Fujimoto, S. Matsuda, N. Takahashi and I. Saito, J. Am. Chem.
Soc., 2000, 122. 5646; K. Fujimoto, N. Ogawa, M. Hayashi, S.
Matsuda and I. Saito, Tetrahedron Lett., 2000, 41, 9437; I. Saito, Y.
Miyauchi, Y. Saito and K. Fujimoto, Tetrahedron Lett., 2005, 46, 97;
Y. Yoshimura, Y. Ito and K. Fujimoto, Bioorg. Med. Chem. Lett.,
2005, 15, 1299; K. Fujimoto, Y. Yoshimura, T. Ikemoto, A.
Nakazawa, M. Hayashi and I. Saito, Chem. Commun., 2005, 3177; M.
Ogino, Y. Yoshimura, A. Nakazawa, I. Saito and K. Fujimoto, Org.
Lett., 2005, 7, 2853; S. Ogasawara and K. Fujimoto, ChemBioChem,
2005, 6, 1756; Y. Yoshimura, Y. Noguchi, H. Sato and K. Fujimoto,
ChemBioChem, 2006, 7, 598.
15 Incorporation of CVU into oligonucleotides was confirmed byenzymatic digestion and MALDI–TOF–MS analysis. MALDI–TOF–
MS: calcd. for ODN 2 (C206H 259 N75O 128P20) [(M–H)–] 6452.25; found6452.60.
16 J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press:
New York, 1989.
17 The rate constant (k) for deamination of C<> CVU photoadduct in theligation site can be calculated from equation (1) where x is thefraction of cytosine converted into uracil which is determined fromthe intensities of the cleaved bands and t is time of incubation.
k = –ln(1–x)/t (1)Kenzo Fujimoto,*ab Shigeo Matsuda,c Yoshinaga Yoshimura,bTakashi Matsumura,a Masayuki Hayashid and Isao SaitoeaSchool of Materials Science, Japan Advanced Institute of Science andTechnology, Ishikawa 923-1292, Japan. Fax: +81 761 51 1671; Tel: +81
761 51 1671; E-mail: kenzo@jaist.ac.jpb
PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012,
Japan
cCurrent address: Department of Chemistry, The Scripps ResearchInstitute, La Jolla, CA 92037, USAd
Department of Synthetic Chemistry and Biological Chemistry, Faculty ofEngineering, Kyoto University, Kyoto 606-8501, JapaneCurrent address: NEWCAT Institute, School of Engineering, Nihon
University, Koriyama 963-8642, Japan

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