Pseudo-dumbbell-type molecular beacon probes bearing modified deoxyuridine derivatives and a silylated pyrene as a fluorophore

Article abstract of DOI:10.1246/bcsj.20140372

We have recently reported a novel pseudo-dumbbell-type molecular beacon probe (Probe 1) possessing polyamine-connected deoxyuridine (U) and silylated pyrene. The probe showed weak fluorescence signal while it stayed alone. Fluorescence signal of the probe was increased in the presence of the complementary DNA. In this study, we prepared new molecular beacons, Probe 2 and Probe 3, possessing the elongated stem portion of Probe 1. In addition, one U in Probe 2 is substituted by anthraquinone-bearing deoxyuridine residue (Y) in Probe 3. Probe 4 is essentially the same as Probe 1 but one deoxyguanosine in the loop portion of Probe 1 is substituted by deoxyinosine in Probe 4. In Probe 5, 3′- terminal deoxycytidine of Probe 3 is substituted by deoxyadenosine. The fluorescence signal of these probes is effectively quenched in the absence of target DNA. Among all, Probe 3 shows the most effective quenching. On the other hand, the signal is substantially increased in the presence of complementary DNA. The ratio of signal to background in case of Probe 3 is the highest. All these probes also recognize single nucleotide alternation in the target DNA to a different extent. The sequence recognition ability of Probe 3 is also the highest among all the probes.

Full text of DOI:10.1246/bcsj.20140372

Pseudo-Dumbbell-Type Molecular Beacon Probes Bearing Modified
Deoxyuridine Derivatives and a Silylated Pyrene as a Fluorophore
Jakir Ahmed Chowdhury, Tomohisa Moriguchi, and Kazuo Shinozuka*
Division of Molecular Science, Graduate School of Science and Technology, Gunma University,
1-5-1 Tenjincho, Kiryu, Gunma 376-8515
E-mail: sinozuka@chem-bio.gunma-u.ac.jp
Received: December 1, 2014; Accepted: January 23, 2015; Web Released: January 30, 2015
We have recently reported a novel pseudo-dumbbell-type molecular beacon probe (Probe 1) possessing polyamine-
connected deoxyuridine (U) and silylated pyrene. The probe showed weak fluorescence signal while it stayed alone.
Fluorescence signal of the probe was increased in the presence of the complementary DNA. In this study, we prepared
new molecular beacons, Probe 2 and Probe 3, possessing the elongated stem portion of Probe 1. In addition, one U in
Probe 2 is substituted by anthraquinone-bearing deoxyuridine residue (Y) in Probe 3. Probe 4 is essentially the same as
Probe 1 but one deoxyguanosine in the loop portion of Probe 1 is substituted by deoxyinosine in Probe 4. In Probe 5, 3¤-
terminal deoxycytidine of Probe 3 is substituted by deoxyadenosine. The fluorescence signal of these probes is effectively
quenched in the absence of target DNA. Among all, Probe 3 shows the most effective quenching. On the other hand, the
signal is substantially increased in the presence of complementary DNA. The ratio of signal to background in case of
Probe 3 is the highest. All these probes also recognize single nucleotide alternation in the target DNA to a different extent.
The sequence recognition ability of Probe 3 is also the highest among all the probes.
Fluorescence-labeled oligonucleotide probes are remarkably
important as a tool in modern life science for medical diag-
nostics and genetic studies.1 To date, there have been many
reports on specific fluorescent oligonucleotide probes, such as
probes, natural nucleobase, guanine and thymine, are used as
3
4,35
33
an internal quencher,
and graphene oxide is used as an
­3
external quencher. The monomer­excimer emission is also used
3
6
to develop this kind of quencher-free molecular beacon.
4­10
molecular beacon types
and other fluorophore­quencher
for the detection of specific genes
Meanwhile, we have developed a novel silylated pyrene
derivative possessing dimethylsilyl function with modifiable
terminal group as a new labeling agent of biological substances
conjugated probes,1
1­13
through the binding to the complementary region of the target.
3
7­39
40
One of the most useful probes for the detection of nucleic acid
such as oligoDNA
and lipids. The compound is more
is the so-called molecular beacon (MB) probe.1
4­20
Molecular
advantageous as a fluorescent labeling agent compared to
original pyrene since it exhibits enhanced (more than 2 times)
fluorescent quantum yield along with a bathochromic shift in
both absorption and emission, due to the Si-associated σ­π
beacon probes undergo a closed to open conformational change
in the absence and presence of target DNA with simultaneous
6
,21­24
characteristic changes in their fluorescent intensities.
4
1,42
Traditional molecular beacon probes form a stem-loop structure
interaction.
Once the derivative is incorporated into the
and are doubly end-labeled with a fluorophore (fluorescence
5¤-terminus of a DNA molecule, however, the fluorescence
signal of the derivative is severely quenched by neighboring
nucleobase including thymine, cytosine and guanine because
donor) and a fluorescence quencher (acceptor).2
5,26
The stem
structure brings the fluorophore and quencher in close prox-
imity allowing energy to be transferred directly from the
fluorophore to the quencher. When the probe meets a target
DNA or RNA molecule, the stem-loop structure is resolved into
a linear probe­target complex that is longer and more stable than
the stem hybrid. As a result of this conformational rearrange-
ment, the fluorophore and the quencher move apart from each
other, restoring fluorescence emission. Since the first inven-
4
3
of the photoinduced electron transfer. Utilizing these unique
features of silylated pyrene, we have developed a novel
quencher-free MB system recently (Probe 1, formerly desig-
4
4
nated GK-2139). The probe has partial self-complementary
sequence and, therefore, it forms pseudo-dumbbell-type sec-
ondary structure. In that form, a silylated pyrene molecule
introduced at the 5¤-terminus of the probe is presumed to be in
the proximal position to a deoxycytidine residue at the 3¤-
terminus. In this study, we have found that the probe gives only
a weak fluorescence signal while it stays alone at near physio-
logical conditions, whereas the signal substantially increases
upon binding to the complementary DNA with the loop por-
4
tion of molecular beacons by Tyagi et al., many approaches
of improvements to design and development of new types of
molecular beacon have been reported.2
7­32
A most recent exam-
ple of such improvements in their design is the development of
quencher-free molecular beacon probes.3
3­36
The quencher-free
4
4
molecular beacons are oligonucleotides which are monolabeled
tion. Thus, the probe possesses target-detecting ability in
terms of its characteristic change on fluorescence signal. The
probe also exhibits certain sequence discriminating ability.
3
3
34
35
in the loop, or in the stem and also multilabeled in the loop,
3
6
or in the stem. In those quencher-free molecular beacon
496 | Bull. Chem. Soc. Jpn. 2015, 88, 496–502 | doi:10.1246/bcsj.20140372
© 2015 The Chemical Society of Japan

U
or
Target DNA
hybridization
Y
U
U
or
Y
G
or
I
U
3
' 5'
5'
A
U C A
or
3'
G
or
I
U
C
or
A
A X
X
Probe 1 to 5
NH2
O
N
H
CH3
NH2
N
N
N
N
O
HN
5'
N
Si
O P O
N
O
O
U=
O
O
O
X=
CH3
O-
H2N
O
O
O
N
O
H
N
N
N
HN
HN
NH
O
O
I=
O
O
O
O
O
N
Y=
O
O
O
Figure 1. Basic structure and principle of molecular beacon probe bearing silylated pyrene at 5¤-terminus.
Table 1. Sequence and Structure of Modified Oligonucleotides
Name
Sequencea),b),c)
Probe 1
Probe 2
Probe 3
Probe 4
Probe 5
ODN-1
ODN-2
ODN-3
ODN-4
3¤-CUTTTAGUTUAAGTACTGTT TTCGGAAA-X-5¤
3¤-CCUTTTAGGUTUAAGTACTG TTTTCGGAAA-X-5¤
3¤-CCUTTTAGGYTUAAGTACTG TTTTCGGAAA-X-5¤
3¤-CUTTTAGUTUAAGTACTGTT TTCGIAAA-X-5¤
3¤-ACUTTTAGTYTUAAGTACTG TTTTCGGAAA-X-5¤
5¤-TGTTTCATGACAAAAGCC-3¤
5¤-TGTTTCATGAGAAAAGCC-3¤
5¤-TGTTTCATGTCAAAAGCC-3¤
5¤-TGTTTCATCACAAAAGCC-3¤
a) U denotes the polyamine bearing deoxyuridine, X denotes the dimethylsilylated pyrene, Y
denotes the anthraquinone bearing deoxyuridine, and I denotes deoxyinosine. b) Italicized letters
indicate the complementary region to ODN-Probe. c) Bold and underlined letters in ODN-2 to -4
indicate the corresponding single-nucleotide alternations to fully matched target DNA (ODN-1).
For further exploration to develop a new fluorescent probe
reported methods.45­47 Incorporation of phosphoramidite deriv-
ative of dimethylsilylated pyrene was carried out in a manner
similar to that described previously. After the assembly, the
support-bound fluorescent oligoDNA was treated with con-
centrated ammonium hydroxide (55 °C, 12 h) followed by
reversed-phase HPLC and gel-filtration as usual.
The sequence of all synthesized molecular beacon probes
(Probe 1 to 5) and their complementary DNA (ODN-1) along
with the partial complementary strands (ODN-2 to -4) having
one base mismatch are shown in Table 1. In the molecular
beacon probes, the silylated pyrene molecule (X) was placed
at the 5¤-terminus neighboring consecutive deoxyadenosine
residues through phosphodiester linkage. In addition, modified
deoxyuridine residues bearing either polyamine molecule (U)
or anthraquinone molecule (Y) at their C-5 position are placed
instead of natural thymidine residues in the stem-forming
region. In all cases, oligoDNA probes were designed by com-
for the detection of specific gene fragments, we have syn-
thesized several new molecular beacon probes (Probes 2
to 5) by modifying their stem portion or substituting certain
deoxyguanosine residue to deoxyinosine residue (Figure 1).
Here, we would like to present the results of our studies of
the fluorescence properties as well as the thermal stabilities of
both the secondary structures of the new probes and the
complexes consisting of the probes and the targets to find the
influence of those modifications to the fluorescence properties
of the probes.
3
7
Results and Discussion
Preparation of Materials. All the modified oligomers
were synthesized using an automated DNA synthesizer
ABI 392). Incorporation of the C-5 modified 2¤-deoxyuridine
(
derivative into the oligomers was carried out using previously
Bull. Chem. Soc. Jpn. 2015, 88, 496–502 | doi:10.1246/bcsj.20140372
© 2015 The Chemical Society of Japan | 497

(a)
(b)
Wavelength/nm
Time/min
Figure 2. (a) Fluorescent spectra of Probe 1 (1.0 ¯M) changes upon addition of ODN-1 in 10 mM sodium phosphate buffer (pH 7.2)
containing 100 mM NaCl at room temperature. The spectra were collected at 5 min intervals after addition of ODN-1 to the solution
of Probe 1. (b) Kinetic curve of fluorescence signal of Probe 1 (1.0 ¯M) at 380 nm with ODN-1 (1.0 ¯M). Excitation wavelength is
350 nm.
prising a self-complementary sequence stem portion and a 15 nt
long loop portion which is complementary to HIV-1 tat/rev
sequence. In this structure, silylated pyrene connected next to
consecutive dA residues will be in a close proximately to dC
residue at 3¤-terminus except Probe 5 (Table 1 and Figure 1).
In Probe 5, 3¤-terminal deoxycytidine of Probe 3 is substituted
by deoxyadenosine residue and, therefore, the corresponding
CG base pair in the stem-forming region is switched to AT base
pair. The structures of the synthesized oligonucleotides were
confirmed by ESI-mass spectrometry (Supporting Information,
Table S1).
Probe 1
Probe 2
Probe 3
Probe 4
Probe 5
Probe 1+ODN-1
Probe 2+ODN-1
Probe 3+ODN-1
Probe 4+ODN-1
Probe 5+ODN-1
Fluorescence Properties of the Probes. Recently, we have
reported a novel stem-loop type MB (Probe 1) possessing
polyamine-connected deoxyuridine and silylated pyrene. The
probe gives only a weak fluorescence signal while it stays alone
in near physiological conditions. Under these conditions, the
probe forms a pseudo-dumbbell-shaped structure (Figure 1)
and observed fluorescence quenching is presumably due to the
photoinduced electron transfer involving the adjacent deoxy-
cytidine (dC) residue in the structure,44 whereas the signal
substantially increased upon binding to form a complex with
the complementary DNA to the loop portion.
Wavelength/nm
Figure 3. Fluorescent spectra of MB probes and complexes
with ODN-1 in 10 mM sodium phosphate buffer (pH 7.2)
containing 100 mM NaCl at room temperature. The con-
centration of each oligonucleotide is 1.0 ¯M. Excitation
wavelength is 350 nm.
At first, we investigated the time dependency of the observed
fluorescence increment of Probe 1 in the same conditions
mentioned above (10 mM sodium phosphate buffer containing
effectively quenched while they stayed alone in the solution.
As shown in Figure 3, however, the extent of the quenching
is varied among the probes. The quenching in Probe 2 and
Probe 4 seems to be slightly less effective than that of the
original Probe 1. The quenching of fluorescence signal in
Probe 3 is found to be the most effective among the probes.
1
00 mM sodium chloride). Thus, the fluorescence signal of
Probe 1 under the presence of ODN-1 was measured in differ-
ent time periods and the results are depicted in Figure 2a. As
shown in the figure, the probe exhibited marked fluorescence
signal at 380 nm upon excitation at 350 nm within a few min-
utes after the addition of ODN-1 in the solution. Figure 2b
summarizes the time-dependent increment of the fluorescence
signal at 380 nm. As it is clear from Figure 2b, the fluorescence
signal increased rapidly and almost reached a plateau within
The observed quantum yield (Φ ) of Probe 3 is 0.002 and the
f
4
4
value is significantly lower than that of Probe 1 (Φ = 0.008),
Probe 2 (Φ = 0.017), and Probe 4 (Φ = 0.015). Since
f
f
f
Probe 3 possesses modified deoxyuridine residue bearing
anthraquinone moiety, a well-known fluorescence quencher,
at its C-5 position, the observed effective quenching in Probe 3
could be attributed due to the additional quenching effect
brought by the anthraquinone moiety in the stem portion. Using
Probe 5 which does not have deoxycytidine at 3¤-terminas, we
tried to estimate the quenching effect of anthraquinone in
1
0 min. In the following experiments, therefore, fluorescence
signal was measured at about 30 min after the mixing of the
probes and the complements.
The florescence spectra of other molecular beacon probes as
well as the complexes with their complement are shown in
Figure 3. In all cases, fluorescence signals of the probes were
498 | Bull. Chem. Soc. Jpn. 2015, 88, 496–502 | doi:10.1246/bcsj.20140372
© 2015 The Chemical Society of Japan

Probe 3. As it is listed in Table 1, the observed quantum yield
of Probe 5 is 0.003 and the value is nearly the same as that
of Probe 3. The result strongly suggests that the effective
quenching in Probe 3 is mainly brought by the anthraquinone
moiety and the effect is greater than that of deoxycytidine, at
least, in the current probes.
The presence of complementary DNA (ODN-1) brought
about the distinct increase in the fluorescence signal of all
probes as those are depicted in Figure 3. The observed incre-
ment of the signal can be attributed to the loss of the secondary
structure of the probes along with the formation of a double
helical complex with ODN-1.44 Although the sequence recog-
nition portions in all the probes are identical, there were slight
differences in the magnitude of their fluorescence signal. The
Φf values of the complexes of Probe 2, Probe 3, Probe 4,
and Probe 5 with ODN-1 were 0.165, 0.120, 0.153, and 0.124
Table 2. Sequence Discrimination Ability of Different MB
Probes
Quantum
yield
Qcomplex/
Qprobe
Qmis/
Qfull
OligoDNA
a)
b)
Probe 1
0.008
0.160
0.102
0.104
0.050
0.017
0.165
0.122
0.125
0.089
0.002
0.120
0.062
0.071
0.049
0.015
0.153
0.115
0.119
0.087
0.003
0.124
0.101
0.088
0.079
®
®
Probe 1 + ODN-1
Probe 1 + ODN-2
Probe 1 + ODN-3
Probe 1 + ODN-4
Probe 2
Probe 2 + ODN-1
Probe 2 + ODN-2
Probe 2 + ODN-3
Probe 2 + ODN-4
Probe 3
Probe 3 + ODN-1
Probe 3 + ODN-2
Probe 3 + ODN-3
Probe 3 + ODN-4
Probe 4
Probe 4 + ODN-1
Probe 4 + ODN-2
Probe 4 + ODN-3
Probe 4 + ODN-4
Probe 5
20.00
12.75
13.00
6.25
®
1.00
0.64
0.65
0.31
®
9.71
7.18
7.35
5.24
®
1.00
0.74
0.76
0.54
®
60.00
31.00
35.50
24.50
®
1.00
0.52
0.59
0.41
®
respectively (Φ for the complex of Probe 1 + ODN-1 is
f
4
4
0
.160 ). Among the complexes, the complex of Probe 2 +
ODN-1 exhibited highest Φ value and that is slightly larger
f
than that of Probe 1. The quantum yield of probe­target
complex of Probe 3 was the lowest among all. On the other
hand, the ratio between the quantum yield of probe itself versus
probe­target complex (background to signal) in Probe 3 was
10.20
7.67
7.93
5.80
®
1.00
0.75
0.78
0.57
®
1
:60 and it was the highest ratio since the ratio for Probe 1,
Probe 2, Probe 4, and Probe 5 are 1:20.0, 1:9.7, 1:10.2, and
:41.3 respectively (Table 2). The data indicate that Probe 3 is
Probe 5 + ODN-1
Probe 5 + ODN-2
Probe 5 + ODN-3
Probe 5 + ODN-4
41.33
33.66
29.33
26.33
1.00
0.81
0.71
0.64
1
a quite sensitive probe reporting the existence of certain gene
fragments in solution through the change of its fluorescence
signal.
a) Ratio of quantum yield of complexes (Qcomplex) with that of
probes alone (Qprobe). b) Ratio of quantum yield of mismatched
complexes (Qmis) with that of matched strand (Qfull).
The Effect of Mismatch in the Target DNA to the Fluo-
rescence Signal of the Probes. The effect of single nucleotide
alteration in the complementary DNA to the fluorescent signal
was also investigated using single mismatched targets, namely
ODN-2 to ODN-4. In the targets, a nucleotide unit near the
middle of the complementary sequence to the probe is altered
from the full-matched target (ODN-1) as those are indicated in
Table 1. The combination of the mismatch includes G/G, T/T,
and C/C. The obtained fluorescence signal of the complexes
consisting of the probes and mismatched targets decreased in
all cases compared to those of the corresponding complexes
consisting of the probes and fully matched target (ODN-1)
as those are shown in Figure 4 and the Figures S-1 to S-3
Probe 3
Probe 3+ODN-1
Probe 3+ODN-2
Probe 3+ODN-3
Probe 3+ODN-4
(Supporting Information). Thus, all probes recognized single
nucleotide alternation in the target in terms of the fluorescence
signal. However, recognition of ODN-4 by the probes seems to
be accomplished most effectively since the decrement of the
fluorescence signal on ODN-4 was more prominent compared
to those on other combination of mismatch in all tested cases.
Recognition of ODN-2 seems to be almost the same or a little
bit worse than the recognition of T/T mismatch using ODN-3.
According to the results, Probe 3 recognizes nicely all
three mismatches since the ratio between the quantum yield of
probe-fully matched target versus probe-mismatched targets in
Probe 3 exhibited smaller values than other cases.
Wavelength/nm
Figure 4. Fluorescent spectra of Probe 3 and complexes
with ODN-1 to -4 in 10 mM sodium phosphate buffer
(pH 7.2) containing 100 mM NaCl at room temperature.
The concentration of each oligonucleotide is 1.0 ¯M.
Excitation wavelength is 350 nm.
The Effect of Deoxyinosine to the Fluorescence Signal of
Probe 4. In the previous study, we found that the Φ value
f
of the complex consisting of Probe 1 and DNA-1 was much
smaller than that of silylated pyrene. It was speculated that this
could be partially reasoned by long-range fluorescence quench-
ing of deoxyguanosine residues near the 5¤-teminus.44 There-
fore, we have substituted the 4th deoxyguanosine residue in
Probe 1 to deoxyinosine in Probe 4 expecting to have larger Φf
Bull. Chem. Soc. Jpn. 2015, 88, 496–502 | doi:10.1246/bcsj.20140372
© 2015 The Chemical Society of Japan | 499

Table 3. UV Melting Temperatures of MB Probesa)
ODN-1
Tm/°C
ODN-2
ODN-3
ODN-4
OligoDNA
Alone
Tm/°C
¦T /°C
m
Tm/°C
¦T /°C
m
Tm/°C
¦T /°C
m
Probe 1
Probe 2
Probe 3
Probe 4
Probe 5
37.3
41.2
45.2
35.8
45.1
51.4
51.3
53.7
49.5
52.9
40.8
42.6
40.6
39.0
43.7
10.6
8.7
13.1
10.5
9.2
43.0
45.7
41.7
40.0
44.7
8.4
5.6
12.0
9.5
8.2
36.9
36.0
36.6
32.8
35.8
14.5
15.3
17.1
16.7
17.1
a) Conditions: UV melting temperature profile measured in 10 mM sodium phosphate buffer containing 100 mM NaCl
and concentration of oligomer was 3 ¯M for each strand. ¦Tm = (Tm value with full matched complement ¹ Tm value
with mismatched complement).
value for Probe 4 after the formation of complex with DNA-1.
However, we could not see any such effect since fluorescent
quantum yield of the complex consisting of Probe 4 and DNA-
recognition of ODN-3 was the lowest. In addition, such
sequence recognition ability seems to be highest in Probe 3
because the obtained ¦Tm values for Probe 3 exhibited the
largest values to all mismatched targets.
1
was almost the same as that of the complex consisting of
Probe 1 and DNA-1 as it is shown in Table 2.
As discussed earlier, quenching of fluorescence signal in
Probe 3 is most effective among the probes while they are
staying alone. Also, recognition of the mismatched target by
the probes in terms of fluorescence signal was most effective
on ODN-4 since the decrement of the fluorescence signal on
ODN-4 compared to ODN-1 was the most prominent among
all the mismatched targets. Thus, there are some correlations
between the fluorescence signal and thermal stability of the
complex formed by the probe and the target. In the cases
described in this study, we found an overall trend that in fluo-
rescence signal of the probe was effectively quenched as the
melting temperature of the probe is increased when the probe
stays alone. However, in the cases of Probe 3 and Probe 5,
anthraquinone moiety affects both the increment of thermal
stability of the probe and the quenching of the fluorescence
signal. In the complex, the complex having larger ¦Tm value
gives smaller fluorescence signal.
Hybridization Abilities of Molecular Beacon-Type Probe.
The UV melting studies were performed to identify the duplex
forming ability as well as the sequence discrimination ability of
the molecular beacon-type probes. The melting temperatures
(
Tm) of the molecular beacon probes as well as the complexes
with their full-matched and single mismatched targets obtained
from the studies are listed in Table 3. As it was reported
4
4
earlier, Tm value of Probe 1 possessing three polyamine
bearing deoxyuridine within the 5-bp stem portion was 37.3 °C
and it is about 10 °C higher than that of the corresponding
oligoDNA possessing natural thymidine residues instead of the
modified deoxyuridine residues. This could be attributed to the
duplex-stabilizing effect of the modified deoxyuridine residues
4
8
in Probe 1. Tm value of Probe 2 possessing three polyamine
bearing deoxyuridine within the longer (6-bp) stem portion was
around 41 °C. Meanwhile, Tm value of Probe 3 was around
4
5 °C and it is about 4 °C higher than that of Probe 2. In
Conclusion
Probe 3, one polyamine bearing deoxyuridine in Probe 2 is
substituted with anthraquinone bearing deoxyuridine. Thus, the
result can be explained in that anthraquinone molecule attached
at C-5 position of deoxyuridine through an alkyl linker inter-
calates into the double helical portion of Probe 3 to increase
the thermal stability.49 In addition, the data indicates that the
duplex-stabilizing effect of anthraquinone is stronger than that
New molecular beacon-type probes bearing modified
deoxyuridine derivatives and a silylated pyrene as a fluoro-
phore were synthesized. The probes have a region of self-
complementary sequence and form pseudo-dumbbell secondary
structure while they stay alone. The modified deoxyuridine
derivatives are located in the stem portion of the struc-
ture and 3¤-terminal deoxycytidine is presumed to be in the
proximal position to silylated pyrene at the 5¤-terminus in the
structure. Under these conditions, fluorescence signals of the
probes are efficiently quenched. The terminal deoxycytidine is
responsible to the quenching, however, anthraquinone moiety
located in the opposite position of silylated pyrene seems to
have greater quenching effect. Also the thermal stability of the
secondary structure depends on the kind of modification on the
modified deoxyuridine residues. On the other hand, the signal
of the probes markedly recovered while they formed complexes
with oligoDNA having fully complementary sequence to their
large loop portion. The signal, however, significantly decreased
while they formed complexes with oligoDNA having mis-
matched sequence, to a different extent. Thus, the all probes
tested in the study possess, more or less, an ability to recognize
one base alternation in the complementary region of the target
of the polyamine, at least in this case. T value of Probe 4 was
m
slightly smaller than that of Probe 1. The sequence of Probe 4
is almost identical to that of Probe 1 except one deoxyguano-
sine residue in the loop portion of Probe 1 is substituted to
deoxyinosine residue (Figure 1). At this moment, the observed
lower stability of Probe 4 compared to Probe 1 is not clear to us.
Probe 5 possessing the same numbers of polyamine bearing
deoxyuridine and anthraquinone bearing deoxyuridine as
Probe 3 exhibited almost the same T value as Probe 3.
m
The Tm values of the complexes containing mismatched
targets decreased compared to those of the complexes contain-
ing full-matched target (ODN-1) in all cases, however, to a
different extent. Based on the data in Table 3, all probes seem
to recognize the existence of mismatch in ODN-4 most nicely
among the tested mismatched targets because the ¦Tm values
for ODN-4 exhibited the largest values. On the other hand,
500 | Bull. Chem. Soc. Jpn. 2015, 88, 496–502 | doi:10.1246/bcsj.20140372
© 2015 The Chemical Society of Japan

in terms of fluorescence signal. The complex with mismatched
Supporting Information
target exhibiting larger decrement of T compared to the com-
Table S1, Figures S1­S3, Schemes S1­S3, 31P NMR chart
m
plex with full-matched target gives smaller fluorescence signal.
Thus the thermal stabilities of the secondary structure of the
probe and the complex with the targets are a part of the factors
influencing the fluorescence signal of the probe in addition to
the effective fluorescence quenching brought by anthraquinone.
of compound 5, 14, and 21 and synthetic procedures of
different prosphoramidite derivatives used in this study are
presented in the Supporting information. This material is
available electronically on J-STAGE.
References
Experimental
1
K. Wang, J. Huang, X. Yang, X. He, J. Liu, Analyst 2013,
General Information. Reactions under anhydrous con-
ditions were carried out under an atmosphere of nitrogen.
Reaction progress was observed by thin layer column chroma-
tography on Merck silica gel 60 F254-precoated plates under
a UV lamp. Column chromatography was performed with
1
38, 62.
2
115.
3
J. Guo, J. Ju, N. J. Turro, Anal. Bioanal. Chem. 2012, 402,
H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke, Y.
3
Urano, Chem. Rev. 2010, 110, 2620.
1
silica gel 60N (neutral, spherical, 63­210 ¯m). H NMR and
4
5
S. Tyagi, F. R. Kramer, Nat. Biotechnol. 1996, 14, 303.
W. Tan, X. Fang, J. Li, X. Liu, Chem.®Eur. J. 2000, 6,
31
P NMR spectra were recorded on a JOEL (JNM-ECA 400)
FT NMR SYSTEM at 400 and 161.8 MHz, respectively, using
tetramethylsilane as internal standard. ESI-MS spectra were
recorded on Perkin-Elmer API-100 ESI-MS Spectrophotom-
eter. UV­vis spectra were taken on a UV-2450 Spectrophotom-
eter (Shimadzu). Fluorescence spectra were recorded on a
F-4500 Fluorescence Spectrophotometer (HITACHI) and a
Fluoromax-4 Spectrofluorometer (HORIBA). Oligonucleotides
were purified by reversed-phase HPLC (PU-2089 plus,
JASCO) fitted with a UV­vis detector (SPD-10A, Shimadzu)
and Chromatopac (C-R8A, Shimadzu) using Wakosil 5 C18
column (Ø 4.6 mm © 250 mm).
1107.
6
2004, 126, 6528.
G. T. Hwang, Y. J. Seo, B. H. Kim, J. Am. Chem. Soc.
7
8
9
T. J. Drake, W. Tan, Appl. Spectrosc. 2004, 58, 269A.
Y. J. Seo, J. H. Ryu, B. H. Kim, Org. Lett. 2005, 7, 4931.
G. Goel, A. Kumar, A. K. Puniya, W. Chen, K. Singh,
J. Appl. Microbiol. 2005, 99, 435.
J. Huang, X. Yang, X. He, K. Wang, J. Liu, H. Shi, Q.
Wang, Q. Guo, D. He, TrAC, Trends Anal. Chem. 2014, 53, 11.
A. Okamoto, K. Tainaka, I. Saito, J. Am. Chem. Soc. 2003,
25, 4972.
A. Okamoto, K. Kanatani, I. Saito, J. Am. Chem. Soc. 2004,
26, 4820.
P. J. Hrdlicka, B. R. Babu, M. D. Sørensen, N. Harrit, J.
Wengel, J. Am. Chem. Soc. 2005, 127, 13293.
1
0
11
1
1
1
2
Oligonucleotides Synthesis.
The oligonucleotides con-
taining C-5 modified 2¤-deoxyuridine and dimethylsilylated
pyrene at the 5¤-terminus were synthesized on an automated
DNA synthesizer (Applied Biosystems ABI-392) using a stand-
ard protocol. The C-5 polyamine and/or anthraquinone bearing
modified 2¤-deoxyuridine analogs were incorporated into the
1
3
1
4
78.
5
T. Matsuo, Biochim. Biophys. Acta, Gen. Subj. 1998, 1379,
1
1
S. A. E. Marras, F. R. Kramer, S. Tyagi, Genet. Anal.:
4
5­47
oligomers in a manner similar to that reported previously.
Biomol. Eng. 1999, 14, 151.
G. Bonnet, S. Tyagi, A. Libchaber, F. R. Kramer, Proc.
The phosphoramidite derivative of dimethylsilylated pyrene
was incorporated at the oligomers’ 5¤-termini using higher
1
6
Natl. Acad. Sci. U.S.A. 1999, 96, 6171.
3
7
concentration of the amidite, as described previously. After
the assembly, the support bound modified oligoDNA was treat-
ed with concentrated ammonium hydroxide at 55 °C for 12 h.
Thereafter the oligomer was purified by reversed-phase HPLC,
ethanol precipitation and Sephadex G-25 gel filtration.
17 D. P. Bratu, B.-J. Cha, M. M. Mhlanga, F. R. Kramer, S.
Tyagi, Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13308.
18 Z.-Q. Cui, Z.-P. Zhang, X.-E. Zhang, J.-K. Wen, Y.-F. Zhou,
W.-H. Xie, Nucleic Acids Res. 2005, 33, 3245.
19 H. H. El-Hajj, S. A. E. Marras, S. Tyagi, E. Shashkina, M.
Kamboj, T. E. Kiehn, M. S. Glickman, F. R. Kramer, D. Alland,
J. Clin. Microbiol. 2009, 47, 1190.
Melting Temperature Analysis. A solution of either 3 ¯M
oligonucleotide or its complex of 3 ¯m of each strand in a
buffer of 10 mM sodium phosphate containing 100 mM NaCl
was heated 90 °C for 3 min, cooled gradually to room temper-
ature, and then used for the melting temperature study. UV
absorbance was measured at 260 nm on a Shimadzu UV-2450
Spectrophotometer equipped with a temperature controller. The
2
0
A. K. Chen, M. A. Behlke, A. Tsourkas, Nucleic Acids Res.
2
008, 36, e69.
1
2
2
2
H. S. Joshi, Y. Tor, Chem. Commun. 2001, 549.
S. A. E. Marras, F. R. Kramer, S. Tyagi, Nucleic Acids Res.
2
002, 30, e122.
N. Dobson, D. G. McDowell, D. J. French, L. J. Brown,
J. M. Mellor, T. Brown, Chem. Commun. 2003, 1234.
J. H. Ryu, Y. J. Seo, G. T. Hwang, J. Y. Lee, B. H. Kim,
Tetrahedron 2007, 63, 3538.
W. Tan, K. Wang, T. J. Drake, Curr. Opin. Chem. Biol.
004, 8, 547.
N. Venkatesan, Y. J. Seo, B. H. Kim, Chem. Soc. Rev. 2008,
7, 648.
H. Du, M. D. Disney, B. L. Miller, T. D. Krauss, J. Am.
2
3
¹
1
rate of heating was 0.1 °C min . T values were determined
m
from the first differentials of the absorbance versus temperature
plot using Igor graphing and data analysis program (Wave
Matrices, Inc.).
Fluorescence Measurements. Fluorescence measurements
were carried out with a F-4500 Fluorescence Spectrophotom-
eter and a Fluoromax-4 Spectrofluorometer in a 3 mL cuvette
with excitation at 350 nm and detection at 360­500 nm. All
measurements were conducted in 10 mM sodium phosphate
containing 100 mM sodium chloride with a concentration of
2
4
2
5
2
2
6
3
2
7
Chem. Soc. 2003, 125, 4012.
28 C. J. Yang, H. Lin, W. Tan, J. Am. Chem. Soc. 2005, 127,
1
.0 ¯M each strand.
12772.
Bull. Chem. Soc. Jpn. 2015, 88, 496–502 | doi:10.1246/bcsj.20140372
© 2015 The Chemical Society of Japan | 501

2
9
R. L. Stoermer, K. B. Cederquist, S. K. McFarland, M. Y.
Sha, S. G. Penn, C. D. Keating, J. Am. Chem. Soc. 2006, 128,
6892.
39 T. Moriguchi, M. Ichimura, M. Kato, K. Suzuki, Y.
Takahashi, K. Shinozuka, Bioorg. Med. Chem. Lett. 2014, 24,
4372.
1
3
0
A. Cazzato, M. L. Capobianco, M. Zambianchi, L.
Favaretto, C. Bettini, G. Barbarella, Bioconjugate Chem. 2007,
8, 318.
T. N. Grossmann, L. Röglin, O. Seitz, Angew. Chem., Int.
Ed. 2007, 46, 5223.
P. Conlon, C. J. Yang, Y. Wu, Y. Chen, K. Martinez, Y.
40 T. Moriguchi, M. Hosaka, A. Yoshinari, A. Ozaki, T.
Takeuchi, K. Shinozuka, Chem. Lett. 2009, 38, 966.
41 S. Kyushin, M. Ikarugi, M. Goto, H. Hiratsuka, H.
Matsumoto, Organometallics 1996, 15, 1067.
42 H. Maeda, Y. Inoue, H. Ishida, K. Mizuno, Chem. Lett.
2001, 1224.
1
3
1
3
2
Kim, N. Stevens, A. A. Marti, S. Jockusch, N. J. Turro, W. Tan,
J. Am. Chem. Soc. 2008, 130, 336.
43 T. Moriguchi, M. Hattori, T. Sekiguchi, K. Shinozuka,
Nucleic Acids Symp. Ser. 2009, 53, 31.
3
3
J. W. Yi, J. Park, N. J. Singh, I. J. Lee, K. S. Kim, B. H.
Kim, Bioorg. Med. Chem. Lett. 2011, 21, 704.
Y. Saito, Y. Shinohara, S. S. Bag, Y. Takeuchi, K.
Matsumoto, I. Saito, Nucleic Acids Symp. Ser. 2008, 52, 361.
M. E. Østergaard, P. Cheguru, M. R. Papasani, R. A. Hill,
P. J. Hrdlicka, J. Am. Chem. Soc. 2010, 132, 14221.
K. Fujimoto, H. Shimizu, M. Inouye, J. Org. Chem. 2004,
9, 3271.
T. Sekiguchi, Y. Ebara, T. Moriguchi, K. Shinozuka,
Bioorg. Med. Chem. Lett. 2007, 17, 6883.
M. Mogi, Md. G. Uddin, M. Ichimura, T. Moriguchi, K.
Shinozuka, Chem. Lett. 2010, 39, 1254.
44 J. A. Chowdhury, T. Moriguchi, K. Shinozuka, Chem. Lett.
2014, 43, 1915.
45 H. Sawai, A. Nakamura, S. Sekiguchi, K. Yumoto, M.
Endoh, H. Ozaki, J. Chem. Soc., Chem. Commun. 1994, 1997.
46 H. Ozaki, A. Nakamura, M. Arai, M. Endo, H. Sawai, Bull.
Chem. Soc. Jpn. 1995, 68, 1981.
47 Y. Sato, T. Moriguchi, K. Shinozuka, Chem. Lett. 2012, 41,
420.
48 M. Matsukura, T. Okamoto, T. Miike, H. Sawai, K.
Shinozuka, Biochem. Biophys. Res. Commun. 2002, 293, 1341.
49 A. T. M. Zafrul Azam, M. Hasegawa, T. Moriguchi, K.
Shinozuka, Bioorg. Med. Chem. Lett. 2004, 14, 5747.
3
4
3
5
3
6
6
3
7
3
8
502 | Bull. Chem. Soc. Jpn. 2015, 88, 496–502 | doi:10.1246/bcsj.20140372
© 2015 The Chemical Society of Japan