Selective fluorescence quenching of the 8-oxoG-clamp by 8-oxodeoxyguanosine in ODN

Article abstract of DOI:10.1016/j.bmcl.2008.12.036

The 8-oxoG-clamp, a specific fluorescent probe for 8-oxo-deoxyguanosine (8-oxo-dG), was incorporated into the oligodeoxynucleotide (ODN) within or at the 3′-end of the purine and the pyrimidine sequences. Based on the UV-melting temperature, the 8-oxoG-cl

Full text of DOI:10.1016/j.bmcl.2008.12.036

Bioorganic & Medicinal Chemistry Letters 19 (2009) 727–730
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Selective fluorescence quenching of the 8-oxoG-clamp
by 8-oxodeoxyguanosine in ODN
Tamer Nasr a, Zhichun Li a, Osamu Nakagawa a,b, Yosuke Taniguchi a,b, Sayaka Ono a, Shigeki Sasaki a,b,
*
a Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
b CREST, Japan Science and Technology Agency, Kawaguchi Center Building, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The 8-oxoG-clamp, a specific fluorescent probe for 8-oxo-deoxyguanosine (8-oxo-dG), was incorporated
into the oligodeoxynucleotide (ODN) within or at the 30-end of the purine and the pyrimidine sequences.
Based on the UV-melting temperature, the 8-oxoG-clamp showed slightly lower stabilizing effects on the
duplexes containing 8-oxo-dG at the complementary site than that with dG. On the other hand, 8-oxo-dG
in DNA was selectively detected by fluorescence quenching of the 8-oxoG-clamp.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 11 August 2008
Revised 3 December 2008
Accepted 6 December 2008
Available online 11 December 2008
Keywords:
20-Deoxy-8-oxoguanosine
Fluorescence
Detection
8-OxoG-clamp
Oligodeoxynucleotide
8-Oxo-deoxyguanosine (8-oxo-dG) is a major oxidatively dam-
aged metabolite of deoxyguanosine (dG) by active oxygen species
(Fig. 1), and causes G/C to T/A transversion mutations in DNA1,2
The 8-oxo-dG level is regarded as an index of the oxidative stress
of cells3 and is believed to have relevance to some diseases4 and
aging.5 Accordingly, analysis of 8-oxo-dG is of great significance,
and a variety of methods have been developed using HPLC-EC6,
HPLC/GC-MS,7 antibodies,8 etc.9
It is also of great importance to detect 8-oxo-dG in DNA, and
such a method using a sequence of reactions was reported.10 Fluo-
rescent nucleobases have been widely used for the detection and in
the structural study of nucleic acids.11 However a fluorescent
probe for the detection of 8-oxo-dG in DNA is lacking. Recently,
we reported the 8-oxoG-clamp as the specific fluorescent probe
for 8-oxo-dG with a high discrimination ability from other nucleo-
sides (Fig. 2).12 The structural analysis by 1H NMR has shown that
the selectivity of the 8-oxoG-clamp is due to the complex formed
involving multiple hydrogen bonds with 8-oxo-dG including the
7N-H of 8-oxo-dG (Fig. 2, (a)).13 We now report that fluorescent
property of the 8-oxoG-clamp and its selective quenching by 8-
oxo-dG are retained in the ODN probes.
DNA automated synthesizer (Scheme 1).14 The recognition prop-
erty of the 8-oxoG-clamp was first evaluated by measuring the
melting temperature (Tm) using DNA 3 and 4 incorporating the 8-
oxoG-clamp in the middle position of the sequence (Table 1).
As already reported, 8-oxo-dG has a comparable effect on du-
plex stabilization with dC and dA (Tm, °C: 53, 52 or 53, 50).15 The
8-oxoG-clamp showed a slightly lower Tm value to 8-oxo-dG than
to dG (Tm, °C: 53 vs 51 or 51 vs 48). These results are inconsistent
with our expectation based on the monomer experiments, in
which the 8-oxoG-clamp showed a higher affinity to 8-oxo-dG
than to dG (Ks Mꢀ1: 2.3 ꢁ 106 vs 2.1 ꢁ 105).12 It was reported that
the parent G-clamp without the N-carbobenzyloxy (CBZ) group in-
creased the Tm value toward dG (DTm + 18 °C) compared to the dG
and 5-methyl-dC base pair.16 Unlike the parent G-clamp, the CBZ
group of the 8-oxoG-clamp might not form a hydrogen bond with
7N-H probably due to steric repulsion between the benzene ring of
O
N
O
N
H
N
7
7
N
HN
H N
HN
H N
O
The diol derivative of the 8-oxoG-clamp (1) was transformed to
the corresponding 50-dimethoxytrityl (DMTr)-protected 30-phos-
phoramidite precursor (2) by a conventional method, which was
incorporated into the oligodeoxynucleotides (DNA3-5) by the
8
8
N
N
2
2
dR
dR
8-Oxo-deoxyguanosine
(8-oxo-dG)
Deoxyguanosine
(dG)
* Corresponding author.
Figure 1. Structures of deoxyguanosine and 8-oxo-deoxyguanosine. dR: b-D
-20-
E-mail address: sasaki@phar.kyushu-u.ac.jp (S. Sasaki).
deoxyribofuranosyl.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.036

728
T. Nasr et al. / Bioorg. Med. Chem. Lett. 19 (2009) 727–730
(a)
(b)
O
O
N
100
O
DNA4
O
N H
N
O
N H
N
N
O
N
H
O
H
80
H
O
N7
7N
O
O
O
DNA3
1
H
H
H
H
N
N
N
N
60
N
N
dR
dR
N
O
O
dR
dR
N
H
N
H
8-oxo-dG
dG
8-oxoG-Clamp
8-oxoG-Clamp
40
20
0
DNA5
Figure 2. Complex structure of 8-oxoG-clamp with 8-oxo-dG determined by 1H
NMR (a). Unfavorable repulsion postulated toward dG (b).12
O
400
450
500
550
600
O
HN
Wavelength (nm)
O
O
NH
N
Figure 3. Fluorescent spectra of DNA (3, 4, 5) incorporating the 8-oxoG-clamp and
the 8-oxoG-clamp monomer (1). Fluorescent spectra were measured using 1 M of
the probe DNA (3, 4, 5) or the monomer 1 in a buffer containing 100 mM NaCl,
l
N
R1O
O
O
10 mM MgCl2, and 10 mM phosphate at pH 7.0, 25 °C, with excitation at 365 nm.
OR2
1: R1=R2=H
2: R1= DMTr
R2=
iPr2NPOCH2CH2CN
ever, DNA5 incorporating the 8-oxoG-clamp at the 30-end formed
duplexes with the same Tm values toward both 8-oxo-dG and dG
(Table 2). In this case, the hydrogen bonds of the 8-oxoG-clamp
to 7N-H of 8-oxo-dG might be so weakened in an aqueous environ-
ment that 8-oxo-dG and dG did not form stable base pairs to affect
the thermal stability of the duplex.
DNA3:
DNA4:
DNA5:
b
a
N
=8-oxoG-clamp
Scheme 1. Synthesis of 8-oxoG-clamp modified oligonucletotides (3–5). Reagents
and conditions: (a) 1—DMTrCl, pyridine, 2—iPr2NP(Cl)OCH2CH2CN, iPr2NEt, CH2Cl2,
0 °C, 1 h (82%); (b) DNA synthesizer, HPLC purification.
We next investigated the fluorescence quenching properties of
the 8-oxoG-clamp using DNA3–5. The fluorescence intensity of
the compounds is sometimes decreased when they are incorpo-
rated into the ODN.11 The fluorescent spectra of the DNA probes
incorporating the 8-oxoG-clamp showed a similar or somewhat
higher intensity compared to the monomer 8-oxoG-clamp
(Fig. 3). When the DNA3 incorporating 8-oxoG-clamp in the
homopurine strand was titrated in a buffer containing 100 mM
NaCl, and 10 mM MgCl2, 10 mM sodium phosphate, fluorescence
quenching was observed in the order of 8-oxo-dG > dA > dG > dC
and dT, although the selectivity was not very high. Interestingly,
when the titration was performed in a low-salt buffer containing
10 mM NaCl and 10 mM sodium phosphate in the absence of
MgCl2, selective quenching was observed for 8-oxo-dG (Fig. 4A).
On the other hand, the 8-oxoG-clamp in the homopyrimidine
strand in DNA4 showed less selective quenching to 8-oxo-dG in
the homopurine DNA3 in a buffer containing 100 mM NaCl,
10 mM MgCl2, and 10 mM sodium phosphate (Fig. 4B). The DNA5
incorporating 8-oxoG-clamp at the 30-end showed selective
quenching to 8-oxo-dG in DNA6 (Fig. 4C). In these cases, the titra-
tion experiments in the low-salt buffer produced similar quench-
ing profiles. These results have indicated that the 8-oxoG-clamp
is useful for the detection of 8-oxo-dG in DNA.
It is reported that photoinduced electron transfer and proton-
coupled electron transfer play an important role for the mecha-
nism of fluorescence quenching of dyes by nucleobases,17 and
quenching efficiency between a fluorescent dye and a nucleobase
depends on their distance.11,18 In the case when nucleobases are
oxidized, the quenching efficiency is in the order of 8-oxogua-
nine > guanine > adenine > thymine > uracil.19 This model for
quenching mechanism may rationalize our previous results that
the fluorescence of the 8-oxoG-clamp was most effectively
quenched with 8-oxo-dG by forming a selective complex with mul-
tiple hydrogen bonds.12 The original G-clamp in DNA3 showed
higher Tm value to dG in DNA4 (48 °C) than to 8-oxo-dG (40 °C) un-
der the low-salt buffer conditions, and displayed less selective
quenching to dG and 8-oxo-dG in the order of their intrinsic
quenching efficiency (Fig. 5). It is clear form the comparison of
the CBZ group and the sugar-phosphate backbone of the comple-
mentary strand. Considering such a steric requirement, it was ex-
pected that the 8-oxoG-clamp might form a complex with 8-oxo-
dG in a sterically non-crowded site at the end of the duplex. How-
Table 1
Tm values of 8-oxoG-clamp modified oligonucleotides (3 and 4)a
DNA3: 5’-d(CTTTCTT-N-TCCTTTCT)-3’
DNA4: 3’-d(GAAAGAA-N-AGGAAAGA)-5’
DNA3, N=
DNA4, N=
8-oxoG-clamp
8-oxo-dG
G
C
A
T
8-oxoG-clamp
8-oxo-dG
G
C
A
T
47
48
51
45
46
45
51
45
46
53
50
45
53
45
47
54
48
47
48
53
56
40
43
43
48
52
45
42
44
54
48
45
48
42
53
44
a
UV-melting profiles measured using 2 lM each of the DNA strand in 10 mM
sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 10 mM MgCl2 at the
scan rate of 0.5 °C minꢀ1 at 260 nm.
Table 2
Tm values of 8-oxoG-clamp modified oligonucleotides (5 and 6)a
DNA6: 5’-d(N -TTTCTTCTCCTTTCT)-3’
DNA5: 3’-d(N’-AAAGAAGAGGAAAGA)-5’
Sequences N, N’
Tm
DNA6 (N = 8-oxo-dG)
DNA5 (N0 = 8-oxoG-clamp)
DNA6 (N = dG)
56
56
DNA5 (N’ = 8-oxoG-clamp)
a
UV-melting profiles measured using 2
lM each of the DNA strand in 10 mM
sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 10 mM MgCl2 at the
scan rate of 0.5 °C minꢀ1 at 260 nm.

T. Nasr et al. / Bioorg. Med. Chem. Lett. 19 (2009) 727–730
729
DNA3: 5’-d(CTTTCTT-
DNA4: 3’-d(GAAAGAA-X-AGGAAAGA)-5’
N = 8-oxodG-clamp
N
-TCCTTTCT)-3’
DNA3: 5’-d(CTTTCTT-X-TCCTTTCT)-3’
DNA6: 5’-d(X-TTTCTTCTCCTTTCT)-3’
DNA5: 3’-d( -AAAGAAGAGGAAAGA)-5’
DNA4: 3’-d(GAAAGAA-N-AGGAAAGA)-5’
N
N = 8-oxodG-clamp
N = 8-oxodG-clamp
A
B
C
100
80
100
80
100
DNA5
DNA4
DNA3
dA, dC,dT
80
60
dA, dC, dT
dG
dA, dC, dT
dG
60
60
dG
40
20
0
40
20
0
40
20
0
8-oxo-dG
8-oxo-dG
450
8-oxo-dG
450 500
400
550
600
400
550
Wavelength (nm)
600
450
500
400
550
600
500
Wavelength (nm)
Wavelength (nm)
Figure 4. Fluorescence quenching of the DNA probes (DNA3, DNA4 and DNA5) containing the 8-oxoG-clamp. The fluorescence spectra were measured using 1 lM each of the
probe DNA and the target strand at pH 7.0, and 25 °C, with excitation at 365 nm. Each curve shows the spectra of the probe DNA alone (blue), the duplex with the target
strand with X = dA, dC, dT (black), dG (green) and 8-oxo-dG (red). The underlined strands represent the fluorescent probe ODN. DNA3 was titrated in a buffer containing
10 mM NaCl, and 10 mM phosphate (A), DNA4 and DNA5 were titrated in a buffer containing 100 mM NaCl, 10 mM MgCl2, and 10 mM phosphate (B and C).
DNA3: 5’-d(CTTTCTT-N-TCCTTTCT)-3’
DNA4: 3’-d(GAAAGAA-X-AGGAAAGA)-5’
In conclusion, we have shown that selective fluorescence
quenching of 8-oxoG-clamp by 8-oxo-dG is retained in the ODN
probe. Although selectivity for 8-oxo-dG over dG is not enough,
these results indicate the potential of the 8-oxoG-clamp as a lead
compound for new analytical systems to determine 8-oxo-dG in
DNA. For the more selective and sensitive detection of 8-oxo-dG
in DNA, the development of new 8-oxoG-clamp derivatives is
now in progress.
=G-clamp
100
DNA3(G-clamp)
80
Acknowledgments
60
dG
We are grateful to the support by a Grant-in-Aid for Scientific
Research (A) and Young Scientists (B) from the Japan Society for
the Promotion of Science (JSPS).
40
20
References and notes
8-oxo-dG
0
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