Novel fluorescent oligoDNA probe bearing a multi-conjugated nucleoside with a fluorophore and a non-fluorescent intercalator as a quencher

Article abstract of DOI:10.1016/j.bmcl.2006.02.016

A set of 15mer linear oligoDNA probes bearing a modified nucleoside conjugated with a polyamine/fluorescein/anthraquinone reporting moiety were synthesized. In a single-stranded form, the fluorescence generated by the excitation of fluorescein was efficiently quenched, while marked recovery of the fluorescence was observed when the probes formed duplexes with the fully complementary strand.

Full text of DOI:10.1016/j.bmcl.2006.02.016

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2685–2688
Novel fluorescent oligoDNA probe bearing
a multi-conjugated nucleoside with a fluorophore
and a non-fluorescent intercalator as a quencher
Sinichi Kodama, Satoko Asano, Tomohisa Moriguchi,
Hiroaki Sawai and Kazuo Shinozuka*
Department of Chemistry, Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu 376-8515, Japan
Received 10 October 2005; revised 27 January 2006; accepted 8 February 2006
Available online 9 March 2006
Abstract—A set of 15mer linear oligoDNA probes bearing a modified nucleoside conjugated with a polyamine/fluorescein/anthra-
quinone reporting moiety were synthesized. In a single-stranded form, the fluorescence generated by the excitation of fluorescein was
efficiently quenched, while marked recovery of the fluorescence was observed when the probes formed duplexes with the fully com-
plementary strand.
Ó 2006 Elsevier Ltd. All rights reserved.
Fluorescent labeling of oligonucleotides has been
attracting wide attention over the last decade because
of its ease of handling and relative inexpensiveness com-
pared to the classical radio-labeling method. Many ef-
forts have been focused on the development of a
fluorescent oligonucleotide probe which is able to detect
a specific oligonucleotide sequence in homogeneous
solution. To fulfill this purpose, however, the probe
should possess certain properties such as changing its
fluorescent properties upon hybridization with its com-
plementary strand. Specific pyrene-conjugated oli-
goDNA and oligoRNA probes developed by Yamana
et al.1 and a ‘molecular beacon’ type of probe developed
by Tyagi et al.2 are typical examples of such probes. In
the latter case, particularly, the change of the fluorescent
property relies on the change of the efficiency of energy
transfer (ET) between two different functional groups,
namely, a fluorophore (donor) and a quencher (accep-
tor). The groups are generally attached at both ends of
a probe oligomer, independently, which is not always
an easy task. In addition to this, the probe should have
a stem–loop type of structure prior to hybridize the tar-
get to maintain efficient energy transfer between the two
functional groups. Thus, the method also relies on the
change of the secondary structure of the probe itself
upon hybridization.
Meanwhile, we have previously reported the synthesis of
a linear DNA probe incorporating a fluorophore (fluo-
rescein; acceptor) and an intercalator molecule (acridine;
donor) simultaneously at its 50-terminus.3 The probe
exhibited enhanced hybridization ability due to the pres-
ence of the intercalator. Also, the fluorophore-based
fluorescence induced by the excitation of the intercalator
was strongly quenched upon the hybridization of the
probe to its complementary strand, presumably due to
the cancellation of fluorescent energy transfer (FRET)
from the intercalator to the fluorophore.3
Along with the study to develop further a feasible oli-
goDNA probe, we have designed a novel oligoDNA
probe. In the probe, a modified pyrimidine nucleoside
bearing a fluorophore (fluorescein; donor) and a non-
fluorescent intercalator (anthraquinone; quencher)
molecules at the C-5 position through an appropriate
polyamine molecule is incorporated in the middle of
the sequence (Probe-1 and Probe-2, Fig. 1). Contrary
to the previous probe described above, the current probe
is designed to emit a fluorescent signal upon the hybrid-
ization to its complementary strand. Through the study,
we have found that the fluorescence induced by the exci-
tation of the fluorophore of the probe was efficiently
quenched in the absence of the complement. The fluores-
Keywords: Fluorescent probe; Sequence discrimination; Quenching.
*
Corresponding author. Tel.: +81 277 30 1320; fax: +81 277 30
1321; e-mail: sinozuka@chem.gunma-u.ac.jp
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.016

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S. Kodama et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2685–2688
on the structure of the linker molecule connecting the
Probe-1 5'-TCG TCG CXG TCT CCG-3'
fluorophore and intercalator molecules. Here, we would
like to report the synthesis and properties of a novel
15mer DNA probe conjugated with a multivalent poly-
amine bearing fluorescein and anthraquinone moieties.
Probe-2 5'-TCG TCG CYG TCT CCG-3'
S
H
O
N
N C NH
H
HN
N
O
O
O
N
COOH
O
The synthetic procedure of the probe is summarized in
Scheme 1. In brief, 50-protected 5-(cyanomethoxycar-
bonylmethyl)uridine5 (1) was reacted with the
tris(2-aminoethyl)amine derivative of anthraquinone
(2), readily prepared by the reaction of the amine and
1-chloroanthraquinone, to give the modified nucleoside
(3). The primary amine function of the nucleoside was
reacted with either trifluoroacetic acid ethyl ester or
Fmoc-protected b-alanine to give the corresponding
nucleosides 4a and 4b, respectively. The obtained
nucleosides 4 were further converted to the correspond-
ing phosphoramidite derivatives (5a and 5b) in the usual
manner6 and utilized for the oligomer synthesis with
automated DNA synthesizer (ABI-392).
O NH
X =
O
O
HO
O
(donor)
O (quencher)
S
H
N
H
O
N C NH
H
N
HN
N
O
O
O N
O
COOH
O
O
Y =
NH
O
O
HO
O
(donor)
O
(quencher)
hν
hν
hybridization
ET
After the assembly, the resulting oligoDNAs were
deprotected by treatment with concd NH4OH and then
purified by HPLC using reversed-phase column to give
the precursors (Probe-10 and Probe-20) of the probes.
It should be noted that prolonged coupling period
(350 s) was applied for 5 during the oligomer assembly.
The purified precursors were dissolved in sodium car-
bonate buffer (pH 9.8) and reacted with FITC for 1
day at room temperature.
hν
(quencher)
(donor)
Figure 1. The structure and the sequence of multifunctional fluores-
cent probe bearing a fluorophore and an intercalator.
cence was, however, markedly recovered upon the for-
mation of a double strand with its complementary
strand.4 In addition to these, the probe exhibited certain
differential response with its full-matched and one-base
mismatched complements on both thermal and spectro-
scopical properties. However, these properties depend
Removal of the excess amount of FITC from the reac-
tion mixture with Sephadex G-25 column followed by
purification with reversed-phase HPLC gave the multi-
functional oligoDNA probes (Probe-1 and Probe-2).
H2N
NH2
H
H
O
N
N
O
N
N
N
O
N
N
NH2
HN
OCH2CN
HN
N
N
NH
R
HN
O
N
O
O
TFA ehtyl ester
or
O
O
O
O
O
DMTO
DMTO
O
O
H
DMTO
N
H
O
O
O
O
2
Fmoc-β-alanine + DCC
O
OH
OH
DMA
OH
1
3
4a and 4b
H
N
H
N
O
H
O
N
H
H
O
N
N
NH2
or
N
N
NH2
HN
HN
N
HN
R
N
N
P O
N
O
O
O
O
O
O
N
O
O
O
O
DMTO
O
O
oligomer assembly
then deprotection
Cl
NH
O
O NH
O
NH
O
CN
O
O
O
O
O
N P O
CN
Probe-2'
Probe-1'
5a and 5b
S
S
H
H
H
O
O
N
N
H
C NH
N
N
N C NH
H
CF
3 (4a and 5a)
(4b and 5b)
HN
HN
R=
N
N
O
O
O
O
O
N
O
N
O
O
O
O
COOH
FITC
COOH
O
O
NH
O
O NH
or
H
O
O
O
O
O
HO
N
HO
O
O
O
O
Probe-1
Probe-2
Scheme 1. Synthesis of multifunctional fluorescent oligoDNA probe.

S. Kodama et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2685–2688
2687
The structure of the probes was confirmed by enzymatic
digestion and ESI-mass spectrometry.7
more feasible hybridization ability as well as sequence
discrimination ability compared to Probe-1.
At first, the hybridization ability as well as the sequence
specificity of the probes was examined by UV-melting
experiments under near-physiological conditions (pH
7.2, 0.1 M NaCl) and the obtained Tm values are listed
in Table 1.
Next, the fluorescent properties of the probes were
examined under the same condition as the Tm experi-
ments. After the annealing procedure, the probes were
irradiated at the wavelength corresponding to the
absorption maximum of the fluorescein moiety
(494 nm) in the presence or absence of complementary
strands at 20 °C. The obtained fluorescence spectra are
depicted in Figure 2.
As is shown in Table 1, the obtained Tm values indicate
that the probes retain almost the same or slightly lower
degree of hybridization ability compared to the parental
unmodified oligoDNA (ODN-N) towards the full-
matched complement despite the fact that they have
an intercalative molecule. The magnitude of the Tm dec-
rement in the current probes is, however, less prominent
than in the case of the analogous oligoDNA probes car-
rying fluorescein and an intercalator (pyrene) moieties in
the backbone portion. In such probes, Tm value de-
creased from 6 to 11 °C compared to that of the unmod-
ified control.4b The results suggest that in the current
probes, the anthraquinone moiety brings about the du-
plex-stabilizing effect by the intercalation, although,
the effect is counterbalanced by the introduction of
bulky fluorescein moiety, maybe due to the steric effect
of the moiety. This notion could be supported by the
fact that the Tm values of the corresponding precursors
(Probe-10 and Probe-20) lacking the fluorescein moiety
are much higher than those of the probes (Table 1). Fur-
thermore, the Tm value of Probe-2 bearing an extended
linker portion between the intercalator and the fluoro-
phore is higher than that of Probe-1. These results indi-
cate that the distance between the intercalator and the
bulky fluorescein molecule affects the hybridization abil-
ity in the probe DNA. The factor also affects sequence
discrimination ability of the probes, at least in our
study. For example, the results in Table 1 revealed that
both Probe-1 and Probe-2 retain some sequence discrim-
ination ability since the Tm values of the probes with
mismatched complement are lower than those with
full-matched complement. The decrement of the Tm val-
ues of Probe-2 with mismatched complements is, howev-
er, almost the same as that of unmodified ODN-N and is
considerably larger than that of the Probe-1. The results
indicate that the modified probes retain sequence dis-
crimination ability depending on the distance between
the intercalator and the fluorophore. These features
make Probe-2 more desirable than Probe-1 since it has
As it is clearly shown in Figure 2, both probes exhibited
minimal fluorescence in the absence of the complements
as well as in the presence of non-complementary dT-
15mer (blue and purple lines, respectively). This back-
ground emission, however, seems to be slightly higher
in Probe-1. The observed minimal fluorescence under
the conditions is presumably due to the intramolecular
energy transfer from the fluorescein moiety (kEm
:
515 nm) to the anthraquinone moiety (kmax: 520 nm).
On the other hand, marked fluorescence was detected
in the presence of the full-matched complementary
strand in both probes (black lines). The intensity of
the observed fluorescence compared to that of the
probes existing alone was, however, more prominently
increased in Probe-2. As mentioned above, the anthra-
quinone moiety in the probe would intercalate to the du-
plex composed of the probe and the full-matched
complementary strand upon the hybridization. Such
a
b
+ full-match
+ mismatch-1
+ mismatch-2
alone
+ full-match
+ mismatch-1
6
6
3
0
+ mismatch-2
alone
+ dT-15mer
+ dT-15mer
3
0
500
550
600
500
550
600
Wavelength (nm)
Figure 2. Fluorescent spectra of Probe-1 (a) and Probe-2 (b). The
spectra were measured under the same conditions as the UV-melting
experiments.
Table 1. Tm values (°C) of the duplexes consisting of the probe oligonucleotides and their complementsc
+ Mismatched complement-1a (DTm
)
+ Mismatched complement-2a (DTm
b
b
)
Probes
+ Full-matched complement
ODN-N
Probe-10
Probe-20
Probe-1
Probe-2
63.1
70.0
71.5
61.0
63.2
55.9 (ꢀ7.2)
—
—
56.8 (ꢀ6.3)
—
—
58.0 (ꢀ3.0)
55.9 (ꢀ7.3)
57.9 (ꢀ3.1)
55.3 (ꢀ7.9)
a Sequence of the mismatched complement is as follows in which the underlined position indicates mismatch base; mismatched complement-1, 50-
CGGAGACTGCGACGA-30; mismatched complement-2, 50-CGGAGACGGCGACGA-30.
b DTm = (Tm value with full-matched complement) ꢀ (Tm value with mismatched complement).
c Tm values were determined by computer fitting of the first derivative of UV-melting profile measured in 10 mM of sodium phosphate buffer (pH 7.2)
containing 100 mM of NaCl and 2 lM of each oligonucleotide.

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S. Kodama et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2685–2688
In conclusion, we have successfully synthesized a set of
0.2
0.1
0
a
b
0.2
0.1
0
novel oligoDNA probes bearing a modified nucleobase
conjugated with a polyamine/fluorescein/anthraquinone
reporting moiety. In the probes, Probe-2 bearing an
extended linker portion between the intercalator and
the fluorophore exhibited almost the same hybridization
ability as well as sequence discrimination ability as the
parental unmodified oligoDNA. The probe gave a fluo-
rescent signal depending on the presence or absence of
the complement. The intensity of the signal is also sensi-
tive to the sequence of the complement. These features
are quite desirable for oligoDNA probe to realize an
easy detection of the target sequence in homogeneous
solution.
400
450
500
550
400
450
500
550
Wavelength (nm)
Figure 3. UV-absorption spectra of Probe-1 (a) and Probe-2 (b) in the
absence (blue lines) and in the presence (purple lines) of the
complement. The spectra were measured under the same conditions
as the UV-melting experiments.
References and notes
intercalation lowers the efficiency of the intramolecular
energy transfer from the fluorescein moiety to the
anthraquinone moiety causing the increase of the fluo-
rescent intensity.4a
1. (a) Yamana, K.; Ohashi, Y.; Nunota, K.; Nakano, H.
Tetrahedron 1997, 153, 4265; (b) Yamana, K.; Iwase, R.;
Furutani, S.; Tsuchida, H.; Zako, H.; Yamaoka, T.;
Murakami, A. Nucleic Acids Res. 1999, 27, 2387; (c)
Yamana, K.; Iwai, T.; Ohtani, Y.; Sato, S.; Nakamura, M.;
Nakano, H. Bioconjugate Chem. 2002, 13, 1266; (d)
Nakamura, M. Y.; Fukunaga, Y.; Sasa, K.; Ohtoshi, Y.;
Kanaori, K.; Hayashi, H.; Nakano, H.; Yamana, K.
Nucleic Acids Res. 2005, 33, 5887.
2. (a) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303;
(b) Tyagi, S.; Baratu, D. P.; Kramer, F. R. Nat. Biotechnol.
1998, 16, 49; (c) Piatek, A. S.; Tyagi, S.; Pol, A. C.; Telenti,
A.; Miller, L. P.; Kramer, F. R.; Alland, D. Nat. Biotechnol.
1998, 16, 359; Kostrikis, L. G.; Tyagi, S.; Mhlanga, M. M.;
Ho, D. D.; Kramaer, F. R. Science 1998, 279, 1228.
3. Shinozuka, K.; Seto, Y.; Sawai, H. Chem. Commun. 1994,
1377.
The influence of the sequence of the complements to the
fluorescent properties was also examined. In the pres-
ence of the complement having the one-base mis-
matched sequence used in the Tm study, both Probe-1
and Probe-2 gave decreased fluorescent intensity (green
and red lines, respectively) compared to those of the
probes in the presence of the full-matched complementa-
ry strand. Thus, the fluorescent signal of the probes is
sensitive to the sequence of the target even at 20 °C, con-
trary to the analogous probe reported previously.4a
Also, the results are somewhat consistent with the re-
sults obtained in Tm studies.8
4. (a) Ranasinghe, R. T.; Brown, L. J.; Brown, T. Chem.
Commun. 2001, 1480; (b) Yamane, A. Nucleic Acids Res.
2002, 30, e97.
Interestingly, the UV-absorption spectra of the probes
in the presence of the complement (purple lines)
exhibited hyperchromic effect around the absorption
maximum of fluorescein moiety (494 nm) as those that
are shown in Figure 3. The effect is more prominent
in Probe-2 than in Probe-1. A slight blue shift effect
around the absorption of anthraquinone moiety
(520 nm) was also observed for both probes, suggest-
ing the intercalation of anthraquinone moiety in the
presence of the complement. The exact mechanism
that brings about the observed hyperchromic effect is
not clear at this moment. However, the phenomenon
would, at least, partially be responsible for the fluores-
cent property of the probes, since such efficient
absorption of photon by fluorescein moiety, particu-
larly in Probe-2, would bring about efficient emission
of the moiety, presumably.
5. (a) Shinozuka, K.; Umeda, A.; Aoki, T.; Sawai, H.
Nucleosides Nucleotides 1998, 17, 291; (b) Kohgo, S.;
Shinozuka, K.; Ozaki, H.; Sawai, H. Tetrahedron Lett.
1998, 39, 4067.
6. 31P NMR (CDC13, d); 148.7 and 149.1 (doublet) for 5a
bearing the trifluorocarbonyl function. 148.6. 149.0 (dou-
blet) for 5b bearing the Fmoc function.
7. The average molecular weights calculated form ESI-mass
spectral data are as follows. Probe-1; calcd mass, 5276.6;
obsd mass, 5278.7; Probe-2; calcd mass, 5347.7; obsd mass,
5349.8.
8. The fluorescent intensity of Probe-2 with the other one-base
mismatched sequence (50-CGGAGACAGCGGCGA-30
where the underlined position indicates the mismatch
position, Tm = 59.0 °C) was almost of the same extent as
Probe-2 and full-matched complementary strand (data not
shown).