Catalytic repair of a thymine dimer in DNA via carbazole nucleoside

Article abstract of DOI:10.1246/cl.2006.386

We report the catalytic repair of a thymine dimer incorporated in a DNA duplex via oligodeoxynucleotide (ODN) containing carbazole nucleoside (K). The occurrence of an electron transfer between K and thymine dimer is evidenced by fluorescence quenching me

Full text of DOI:10.1246/cl.2006.386

386
Chemistry Letters Vol.35, No.4 (2006)
Catalytic Repair of a Thymine Dimer in DNA via Carbazole Nucleoside
Yoshinaga Yoshimura and Kenzo Fujimotoꢀ
School of Materials Science, Japan Advanced Institute of Science and Technology and PRESTO,
Japan Science and Technology Agency, 1-1 Ashahidai, Nomi 923-1292
(Received February 1, 2006; CL-060139; E-mail: kenzo@jaist.ac.jp)
We report the catalytic repair of a thymine dimer incorporat-
ed in a DNA duplex via oligodeoxynucleotide (ODN) containing
carbazole nucleoside (K). The occurrence of an electron transfer
between K and thymine dimer is evidenced by fluorescence
quenching measurements. K acts as a good electron donor for
the catalytic repair of a thymine dimer.
Considerable environmental damage to DNA is caused by
the formation of UV-induced photolesions, which can be both
mutagenic and carcinogenic. UV irradiation of cells induces a
[2 þ 2] cycloaddition of pyrimidines located above each other
in the DNA double strand.1 DNA photolyase selectively recog-
nizes the thymine dimer in DNA single and double strands and
repairs it by photoinduced electron transfer, using reduced fla-
vine coenzyme as the electron donor.2 By using a DNA assay
consisting of an artificial DNA base with a flavine structure as
the electron donor, Carell et al. could show that the thymine di-
mer in DNA repairs through reductive photoinduced electron
transfer.3 Barton et al. have observed the repair of a thymine di-
mer as its radical cations with a rhodium intercalator or a naph-
thalene diimide intercalator, which both remove a light-induced
electron from the DNA strand.4 Although a photolyase mimic
composed of small-molecule recognition units was used to
achieve catalytic repair of a thymine dimer,5 the catalytic repair
of a thymine dimer incorporated in a DNA duplex has not been
extensively investigated. The catalytic repair is convenient and
practical from a medicinal perspective. Carbazole derivatives
have strongly hydrophobic surfaces and have been used as elec-
tron donors.6 Deoxyribosides of carbazole have been incorporat-
ed into oligodeoxynucleotide (ODN) and were used as probes to
detect nucleic acid hybridization.7 These properties of carbazole
derivatives are expected to be exploited as electron donors to
study the repair of a thymine dimer in DNA. We have been
studying artificial DNA bases as a tool for photochemical
DNA manipulations.8 We now report on the catalytic repair of
a thymine dimer incorporated in a DNA duplex via carbazole
nucleoside (K).
Scheme 1. Reagents and conditions: (a) KOH, TDA-1, chloro-
sugar, CH3CN, rt, 20 min, 59%; (b) NaOCH3, CH3OH, rt, 2 h,
86%; (c) 4,40-dimethoxytrityl chloride, DMAP, pyridine, rt,
15 h, 36%; (d) (i-Pr2N)2PO(CH2)2CN, 1H-tetrazole, CH3CN,
rt, 2 h, quant.
5. ODNs containing K were characterized by the nucleoside
composition and MALDI-TOF-MS.9 Thymine dimer formation
in synthetic ODNs was performed photochemically according
to a method reported in the literature.10
We determined the feasibility of the photochemical repair of
a thymine dimer in DNA via ODN containing K. When ODN(K)
was irradiated at 365 nm for 30 min in the presence of
ODN1(T=T) 50-d(ATGTGAT=TGCGTGACAGT)-30, we ob-
served the appearance of a peak of ODN1(TT) 50-d(ATGTGA-
TTGCGTGACAGT)-30 in 92% yield as determined by HPLC
with the disappearance of ODN1(T=T) (Figure 1).11,12 MALDI-
TOF-MS indicated that isolated ODN1(TT) obtained from
HPLC purification was a repaired product of ODN1(T=T)
(m=z: calcd 5570.69 for ½M þ Hꢁþ, found 5570.70). The isolated
ODN1(TT) was digested with AP and P1 nuclease at 37 ꢂC for
The synthesis of the phosphoramidite of K is outlined in
Scheme 1. Compound 2 was synthesized from carbazole 1 and
Hoffer’s ꢀ-chlorosugar. Deprotection of 2 with sodium methox-
ide afforded 3 (K) in improved yield as compared with the meth-
od reported in the literature.7 Compound 3 was dimethoxytri-
tylated, and converted into the nucleoside phosphoramidite 5.
The assignments of ꢁ-stereochemistry at C10 for 3 was based on
COSY and NOESY spectra, which showed a cross-peak between
H10 and H40. The modified ODN containing K, ODN(K) 50-
d(ACTGTCACGCKTCACAT)-30, ODN(KK) 50-d(ACTGTCA-
CGCKKTCACAT)-30, ODN(KA) 50-d(ACTGTCACGCKAT-
CACAT)-30, were prepared, according to the standard phosphor-
amidite chemistry, on a DNA synthesizer using phosphoramidite
Figure 1. HPLC analysis of the irradiated ODN(K) in the pres-
ence of ODN1(T=T): (a) before irradiation; (b) irradiated at
365 nm for 30 min; (c) nonirradiated authentic ODN1(TT).
Copyright ꢀ 2006 The Chemical Society of Japan

Chemistry Letters Vol.35, No.4 (2006)
387
Figure 3. Effect of temperature on turnover for three ODNs
containing K.
Figure 2. (a) Fluorescence spectra of ODN(K) excited at
330 nm in the absence/presence of ODN1(T=T) of varying
concentrations: a) 0, b) 0.2, c) 0.4, d) 0.6, e) 0.8, f) 1.0, g) 1.2,
and h) 1.4 equiv. (b) Fluorescence quenching of ODN(K) with
ODN1(T=T) or ODN2(T=T).
turnovers even at 0 ꢂC.
In conclusion, we demonstrated the catalytic repair of a thy-
mine dimer in DNA via ODN containing K. When ODN contain-
ing K was photoirradiated in the presence of ODN containing
thymine dimer, the thymine dimer in DNA was catalytically re-
paired through reductive photoinduced electron transfer. ODN
containing K can be used for the catalytic repair of a thymine di-
mer and has the potential to allow spectroscopic investigation of
electron transfer in DNA.
4 h. Enzymatic digestion of isolated ODN1(TT) showed the for-
mation of dC, dG, dT, and dA in a ratio of 2:6:6:4. The quantum
yield for the photochemical repair by using ODN(K) was esti-
mated (ꢀ ¼ 0:014) at 365 nm, based on the disappearance of
ODN1(T=T) by employing valerophenone as an actinometer.13
When ODN(KK) or ODN(KA) was used in the repair of a thy-
mine dimer, we observed the appearance of a peak of ODN1(TT)
in 94 and 93% yields, respectively. The thermal stability of
the duplex between ODN containing K and ODN1(T=T) was
investigated by monitoring the melting temperature (Tm). The
Tm value (53.0 ꢂC) of ODN(K) and ODN1(T=T) was lower than
that of ODN(K) and ODN1(TT) (56.9 ꢂC), whereas the Tm value
(53.5 ꢂC) of ODN(KK) and ODN1(T=T) was higher than that of
ODN(KK) and ODN1(TT) (51.7 ꢂC). The Tm value (53.6 ꢂC) of
ODN(KA) and ODN1(T=T) was equal to that of ODN(KA) and
ODN1(TT).
To elucidate the electron-transfer phenomena from ODN(K)
to ODN1(T=T), fluorescence quenching of ODN(K) with
ODN1(T=T) was performed in a 50 mM sodium cacodylate buf-
fer (pH 7.0) and 100 mM sodium chloride at a strand concentra-
tion of 200 mM. As shown in Figure 2a, the fluorescence of
ODN(K) was quenched efficiently by ODN1(T=T). On the other
hand, when ODN composed of mismatch bases, ODN2(T=T)
50-d(GCACAGT=TATACAGAGAG)-30, was used as a quench-
er, the fluorescence of ODN(K) was scarcely quenched
(Figure 2b). Furthermore, when ODN2(T=T) was used in repair,
the repaired product of ODN2(T=T) was scarcely observed.
When ODN1(T=T) was irradiated at 365 nm in the presence
of complementary ODN 50-d(ACTGTCACGCAATCACAT)-
30, the repaired product of ODN1(T=T) was scarcely observed.
From these results, ODN(K) can promote the repair of thymine
dimer incorporated in a DNA duplex by electron transfer from
carbazole.
References and Notes
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MALDI-TOF-MS: m=z: calcd 5147.49 for ODN(K) [ðM þ HÞþ],
found 5147.08; calcd 5492.78 for ODN(KK) [ðM þ HÞþ], found
5492.93; calcd 5460.70 for ODN(KA) [ðM þ HÞþ], found 5460.34.
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11 The reaction mixture (total volume 30 mL) containing ODN(K) and
ODN1(T=T) (each 20 mM, strand concn) in 50 mM sodium cacody-
late buffer (pH 7.0) and 100 mM sodium chloride was irradiated with
a 150 W short arc metal halide lamp (1000 mW/cm2) equipped with
a 365 nm UV filter at 0 ꢂC for 30 min.
12 The yield was calculated based on ODN1(T=T).
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15 The reaction mixture (total volume 30 mL) containing ODN(K)
(2.0 mM, strand concn) in the presence of ODN1(T=T) (20 mM,
strand concn) in 50 mM sodium cacodylate buffer (pH 7.0) and
100 mM sodium chloride was irradiated with a 25 W transilluminator
(5.7 mW/cm2) equipped with a 365 nm UV filter at 0 or 25 ꢂC for
12 h.
We determined the feasibility of the catalytic repair of a
thymine dimer in DNA via the modified ODN containing K.
To measure turnover with ODNs containing K, we compared
the number of equivalents of yield to moles of ODN containing
K.14 When ODN containing K was irradiated at 365 nm for 12 h
in the presence of ODN1(T=T), we observed the number of
turnovers by HPLC analysis.15 These results showed that for
all three cases at 25 ꢂC adequate amounts of turnover were ob-
served (Figure 3). In particular, the case of ODN(KK) was the
most efficient of all, yielding 6.7 turnovers. The temperature is
expected to have significant effects, both on the yield of repair
and on turnover efficiency. To test for such effects, we carried
out catalytic repairs at 0 ꢂC. For most cases, we observed the
Published on the web (Advance View) March 11, 2006; DOI 10.1246/cl.2006.386