Hybridization-sensitive fluorescence control in the near-infrared wavelength range

Article abstract of DOI:10.1039/c1ob05252g

A series of near-infrared fluorescent probes were designed based on the concept of emission control caused by interdye excitonic interaction. The fluorescent probes showed very weak emission in the unhybridized state, whereas they emitted near-infrared fl

Full text of DOI:10.1039/c1ob05252g

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Organic &
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PAPER
Hybridization-sensitive fluorescence control in the near-infrared wavelength
range
Shuji Ikeda,a Hiroyuki Yanagisawa,a Akiko Nakamura,a Dan Ohtan Wang,a Mizue Yukia and
Akimitsu Okamoto*a,b
Received 17th February 2011, Accepted 25th March 2011
DOI: 10.1039/c1ob05252g
A series of near-infrared fluorescent probes were designed based on the concept of emission control
caused by interdye excitonic interaction. The fluorescent probes showed very weak emission in the
unhybridized state, whereas they emitted near-infrared fluorescence after hybridization with the
complementary nucleic acid. The hybridization-dependent switching of fluorescence emission made it
possible to monitor mRNA in human cells in the range of near-infrared wavelengths.
with the complementary nucleic acids. The probes made it
possible to monitor mRNA in a human cell in the range of NIR
Fluorescent optical imaging is a rapidly expanding methodology
wavelengths.
Introduction
for the non-invasive evaluation of disease and tumor progression,
with applications in drug and biomarker development.1 The use
of near-infrared (NIR) photons is a promising approach for
biomedical imaging in living tissue.2,3 NIR dyes emit over the
Results and discussion
We focused on the system used in an exciton-controlled
range 650–900 nm, a range in which the absorption coefficients
hybridization-sensitive fluorescent oligonucleotide (ECHO)
of tissues are relatively low, so that the autofluorescence of
probe. The fluorescence of the probe, in which two fluorescent
biological species is minimized. These dyes also have merits,
dyes are attached to a thymine base, is well controlled by excitonic
such as avoiding photobleaching of the fluorophores and using
interaction (Fig. 1).8–10 An excitonic interaction is produced by
inexpensive excitation light sources.4,5 In addition, NIR dyes show
a deeper penetration ability than visible wavelength dyes. The
unique character of NIR dyes is directly reflected in improved
image quality,6,7 and NIR fluorescence imaging has emerged as a
powerful tool for cell and tissue imaging.
It is also important for the establishment of effective nucleic
acid imaging in cells to design an NIR fluorescent probe that
has the feature that the fluorescence is turned off when the
probe does not recognize the target nucleic acid and turned
on when the probe has hybridized the target nucleic acid. The
requirements for NIR fluorescent probes for nucleic acid detection
are not only sequence-selective NIR emission and the avoidance
of non-specific emission, but also expansion of the observable
region of wavelength operated by the new fluorescent probes. The
emission must be polychromatic for the simultaneous monitoring
of different targets.
Herein, we report the synthesis and photophysics of
hybridization-sensitive fluorescence probes with absorption and
emission in the NIR wavelength region. The probes showed
switching of fluorescence emission depending on hybridization
aAdvanced Science Institute, RIKEN, Wako, Saitama, 351-0198, Japan.
E-mail: aki-okamoto@riken.jp; Fax: +81 48 467 9205; Tel: +81 48 467
9238
bPRESTO, Japan Science and Technology Agency, 4-1-8 Honcho,
Kawaguchi, Saitama, 332-0012, Japan
Fig. 1 An exciton-controlled hybridization-sensitive fluorescent oligonu-
cleotide (ECHO) probe.
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Scheme 1 Synthesis of NIR-ECHO probes.
the formation of an H-aggregate between dyes in the probe
and, as a result, emission from the probe before hybridization
is suppressed.11–14 In contrast, dissociation of aggregates by
hybridization with the complementary strand results in disrup-
tion of the excitonic interaction and strong emission from the
hybrid. Multicoloring of the ECHO probe results in the simple
simultaneous detection of multiple target nucleic acid sequences,
and is useful in efforts to understand temporal correlations of gene
expression and interaction between nucleic acids. However, there is
no ECHO probe in which the fluorescence is controlled in the range
of NIR wavelengths. For this context, new nucleotides doubly
labelled with an NIR dye should be designed without loss of a
high on–off performance as a hybridization-sensitive fluorescent
probe based on excitonic interaction chemistry.
A series of new fluorescent nucleotides was synthesized for NIR-
ECHO probes. The coupling of a quinolinium derivative with
the carboxylate-ended alkyl linker 1, prepared by the addition
of 4-methylquinoline to 5-bromovaleric acid, with benzothiazole
or methylquinoline subunits 3, 6 and 9, which were prepared
by extension of the p-conjugate systems of 2, 5 and 8 by
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N,N¢-diphenylformamidine and acetic anhydride, provided fluo-
rescent dyes 4, 7 and 10, respectively (Scheme 1). The carboxylate
end of the alkyl linker of the dyes was activated by succinimidy-
lation, and subsequently two molecules of the activated dyes
were incorporated into an oligodeoxyribonucleotide containing
2¢-deoxyuridine with two amino ends.7 The 4-, 7- and 10-labeled
nucleotides in the synthesized NIR probes were named D644, D662
and D715, respectively.
The absorption and emission properties of the synthesized NIR
probes, Probe1(Dnnn) 5¢-d(CGCAATDnnnTAACGC)-3¢, were inves-
tigated before and after hybridization with the complementary
nucleic acids. The fluorescence emission of these NIR probes was
suppressed in their unhybridized states, but emission appeared
immediately after the probe was added to a solution of the
complementary nucleic acid (Fig. 2). Fluorescence of the hybrid
with the complementary DNA was observed at 655 (Uf 0.29), 674
a lower fluorescence intensity but a longer emission wavelength
than Probe1(D644), which contains a benzothiazole moiety in
the dye. The use of quinoline-based fluorescent dyes may be
effective for the design of the functional dyes with a longer
fluorescence wavelength. The hybridization-sensitive fluorescence
switching is supported by the absorption spectra. The absorption
band of the unhybridized probe appears at about 50, 58 and
70 nm shorter wavelengths for Probe1(D644), Probe1(D662) and
Probe1(D715), respectively, than that of the hybrid. This blue shift
suggested a splitting of the excited state because of the formation of
a bichromophoric H-aggregate. Therefore, the interdye excitonic
interaction in a bichromophoric H-aggregate in the unhybridized
state of the probes suppressed the fluorescence emission of the
probes. This exciton-controlled fluorescence behavior was also ob-
served for the probes with a polythymidine sequence, Probe2(Dnnn
)
5¢-d(TTTTTTDnnnTTTTTT)-3¢. These NIR probes are ECHO
probes that have the characteristic function that hybridization
with the target nucleic acid resulted in a large enhancement of
emission.
(Uf 0.073) and 727 nm (Uf 0.061) for Probe1(D644), Probe1(D662
)
and Probe1(D715), respectively. The fluorescence intensity of the
hybrid was weaker when the probes were hybridized with the
complementary RNA (Uf 0.10, 0.034 and 0.031 for Probe1(D644),
Probe1(D662) and Probe1(D715), respectively), compared with those
of the hybrid with DNA, although the fluorescence was dis-
tinguishable from the very weak fluorescence of unhybridized
The probes formed thermally stable duplexes with the com-
plementary nucleic acids sufficient to detect the fluorescence
emission. For example, the melting temperatures of the duplex
of Probe1(D644), Probe1(D662) and Probe1(D715), and the comple-
mentary DNA were 58, 66 and 54 ◦C, respectively, which were the
same as or higher than that with a natural DNA duplex (54 ◦C),
whereas the melting temperatures of the duplex of Probe1(D644),
Probe1(D662) and Probe1(D715), and the complementary RNA were
41, 38 and 43 ◦C, respectively, which were lower than that with
a natural RNA duplex (43 ◦C). The data on duplex stability
suggests that they reflect the higher fluorescence intensity from
the hybrid with DNA and lower fluorescence intensity from the
hybrid with RNA, as shown in Fig. 2. The duplexes formed by
the probes and their complementary strands showed induced CD
signals with broad negative signals and a sharp positive signal. For
example, the induced CD signals were observed at 550–750 nm
for the duplexes formed by Probe1(D715) and its complementary
DNA (Fig. 3). These signals might result from mixed Cotton
effects derived from several different binding modes of dyes to
DNA, which consist of broad negative Cotton effects at 590 and
640 nm, and a sharp Cotton effect at 665 nm. The results of
Tm and CD measurements suggest that the dyes of Dnnn strongly
interact with the duplex structures after hybridization with the
complementary nucleic acids. The interaction between the dye and
the duplex induces dissociation of a bichromophoric aggregate in
the probe, which contributes to enhancement of the fluorescence
emission.
probes (Uf 0.07, 0.001 and 0.015 for Probe1(D644), Probe1(D662
)
and Probe1(D715), respectively). Probe1(D662) and Probe1(D715),
which have the dyes consisting of only quinoline moieties, showed
Fig. 2 Absorption and emission spectra of NIR-ECHO probes. Absorp-
tion and emission spectra of Probe1(Dnnn) and Probe2(Dnnn) (0.4 mM) were
measured in 50 mM sodium phosphate (pH = 7.0) and 100 mM sodium
chloride. lex = 633 nm for D644 and D662, and 694 nm for D715. Black:
unhybridized probes; red: probes hybridized with the complementary
DNA; orange: probes hybridized with the complementary RNA.
Fig. 3 CD spectrum of the duplex of Probe1(D715) and the complementary
DNA. The spectrum of the duplex (10 mM) was measured in 50 mM sodium
phosphate (pH = 7.0) and 100 mM sodium chloride.
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Development of chemical probes for RNA imaging in living
cells is essential for understanding cell life, which is greatly
desired by life scientists. Having established that the NIR-ECHO
probes are effective for nucleic acid detection, the fluorescence
behavior of the probe was investigated in living cells. The NIR-
ECHO probe capable of binding to the polyA tail of mRNA,
Probe2(D715), was transfected to HeLa cells using a common
transfection reagent, LipofectamineTM 2000, according to the
protocol reported for mRNA imaging using the green-colored
ECHO probe Probe2(D514).10,15,16 After incubation for 1 h and
washing, fluorescence emission was observed through a filter
at about 730 nm. The NIR fluorescence appeared clearly in
transfected cells (Fig. 4). The distribution of NIR fluorescence
JEOL T100LC instrument. DNA was synthesized on an NTS H-6
DNA/RNA synthesizer. Reversed-phase HPLC was performed
on CHEMCOBOND 5-ODS-H columns (10 ¥ 150 mm) with a
Gilson Chromatograph Model 305 using a UV detector Model
118 at 260 nm. MALDI-TOF mass spectra were measured with a
Bruker Daltonics Reflex instrument. UV and fluorescence spectra
were recorded on a Shimadzu UV-2550 spectrophotometer and a
RF-5300PC spectrofluorophotometer, respectively.
1-(4-Carboxybutyl)-4-methylquinolinium bromide (1)
4-Methylquinoline (15.9 mL, 120 mmol) and 5-bromovaleric acid
(18.1 g, 100 mmol) were stirred at 150 ◦C for 5 min under
microwave irradiation (100 W). The reaction mixture was added
to dichloromethane (1 L) with vigorous stirring. After cooling to
room temperature, the solution was sonicated to obtain a white
precipitate. The precipitate was filtered and dried in vacuo. The
white powder was washed with diethyl ether (100 mL), filtered
and dried in vacuo to give 1 as a white powder (11.1 g, 34.2 mmol,
of Probe2(D715) in cells was very similar to that of Probe2(D514
)
bound to mRNA in cells, reported earlier.10,15,16
1
34%): H NMR (DMSO-d6) d 12.07 (br, 1H), 9.42 (d, J = 6.1,
1H), 8.60–8.53 (m, 2H), 8.26–8.23 (m, 1H), 8.08–8.03 (m, 2H),
5.02 (t, J = 7.3, 2H), 3.00 (s, 3H), 2.28 (t, J = 7.3, 2H), 1.99–1.93
(m, 2H), 1.62–1.56 (m, 2H); 13C NMR (DMSO-d6) d 173.9, 158.5,
148.3, 136.7, 135.0, 129.5, 128.9, 127.1, 122.6, 119.3, 56.5, 32.9,
28.8, 21.2, 19.7; HRMS (ESI) calc. for C15H18NO2 ([M - Br]+)
244.1338, found 244.1334.
2-[(E)-2-(N-Acetyl-N-phenylamino)ethenyl]-3-
Fig. 4 Differential interference contrast and epifluorescence image of
Probe2(D715)-transfected HeLa cells. The sample was excited through a
685 15 nm band-pass filter and the fluorescence was collected through a
730 15 nm band-pass filter. Bar: 20 mm.
methylbenzothiazolium iodide (3)
2,3-Dimethylbenzothiazolium iodide (2, 2.91 g, 10 mmol) and
N,N¢-diphenylformamidine (3.92 g, 20 mmol) were suspended in
acetic anhydride (20 mL). The suspension was stirred at 150 ◦C for
1 h. The reaction mixture was cooled to room temperature. Diethyl
ether (150 mL) was added to the reaction mixture with stirring.
The supernatant was discarded by decantation and the residue
was washed with diethyl ether twice and ethyl acetate twice. Ethyl
acetate was added to the residue, and the precipitate was filtered
and dried in vacuo to give 3 as a dark red powder (3.39 g, 7.76 mmol,
78%): 1H NMR (DMSO-d6) d 8.79 (d, J = 14.2, 1H), 8.31 (d, J =
22.0, 1H), 8.09 (d, J = 8.3, 1H), 7.80–7.52 (m, 7H), 5.68 (d, J =
13.7, 1H), 3.87 (s, 3H), 2.05 (s, 3H); 13C NMR (DMSO-d6) d 171.8,
170.0, 144.9, 141.6, 136.8, 130.6, 130.1, 129.0, 128.2, 127.8, 126.5,
124.0, 116.2, 96.4, 35.7, 23.2; HRMS (ESI) calc. for C18H17N2OS
([M - I]+) 309.1062, found 309.1073.
Conclusions
Functional NIR probes for the detection of nucleic acids are
reported in this paper. A series of NIR fluorescent probes were
designed based on the concept of emission control caused by
interdye excitonic interaction, and they contribute to extend the
observable wavelength range operated by ECHO probes. They are
the first NIR probes possessing the function that the absorption
and emission were controlled at various NIR wavelengths, and this
function facilitates the imaging of intracellular mRNA. Although
further aspects remain to be examined in progressing toward an
easier-to-use analysis, such as higher emission intensity for RNA
detection, we anticipate that this new concept of hybridization-
sensitive NIR probes, supported by its photochemical basis, will be
the starting point for the development of a practical NIR detection
in nucleic acid imaging in living organisms.
Fluorescent dye 4
Carboxylic acid 1 (324 mg, 1.0 mmol) and 3 (436 mg, 1.0 mmol)
were suspended in dichloromethane (20 mL). Triethylamine
(1.4 mL, 10 mmol) was added to the suspension and the resultant
solution was stirred at room temperature for 16 h. After the
mixture was concentrated in vacuo, the residue was purified
by silica gel column chromatography (1% acetic acid, 0–20%
methanol–dichloromethane) to give 4 as a dark purple powder
(299 mg, 0.60 mmol, 60%): 1H NMR (DMSO-d6) d 8.39–8.38 (m,
2H), 8.10–8.00 (m, 2H), 7.90 (t, J = 7.8, 1H), 7.82 (d, J = 7.8,
1H), 7.76 (d, J = 7.1, 1H), 7.66 (t, J = 7.7, 1H), 7.50 (d, J = 8.1,
1H), 7.41 (t, J = 7.5, 1H), 7.25 (t, J = 7.5, 1H), 7.03 (d, J = 13.2,
1H), 6.47 (d, J = 12.2, 1H), 4.52 (t, J = 6.7, 2H), 3.70 (s, 3H),
Experimental
General
1H and 13C NMR spectra were measured with a Varian NMR
system 500. Coupling constants (J values) are reported in Hz.
The chemical shifts are shown in ppm, using residual dimethyl
1
sulfoxide (DMSO; d = 2.49 in H NMR, d = 39.5 in 13C NMR)
as an internal standard. ESI mass spectra were recorded on a
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2.29 (t, J = 7.3, 2H), 1.86–1.80 (m, 2H), 1.61–1.55 (m, 2H); 13C
NMR (DMSO-d6) d 174.4, 161.5, 150.1, 143.7, 142.3, 141.8, 137.7,
133.2, 127.5, 126.6, 125.1, 124.6, 124.1, 124.0, 122.4, 117.8, 112.4,
109.4, 109.2, 98.9, 53.6, 33.4, 32.9, 28.3, 21.6; HRMS (ESI) calc.
for C25H25N2O2S ([M - Br]+) 417.1637, found 417.1643.
small amount of dichloromethane. The solution was added to ethyl
acetate (500 mL) with stirring. The supernatant was discarded and
the residue was washed with ethyl acetate twice. Ethyl acetate was
added to the residue and the precipitate was filtered and dried in
vacuo to give 9 as a brown powder (3.94 g, 9.16 mmol, 92%): 1H
NMR (DMSO-d6) d 9.14 (d, J = 6.3, 1H), 8.88 (d, J = 14.2, 1H),
8.36–8.33 (m, 2H), 8.17–8.14 (m, 1H), 7.91–7.84 (m, 2H), 7.71–
7.52 (m, 5H), 6.01 (d, J = 13.7, 1H), 4.45 (s, 3H), 2.07 (s, 3H);
13C NMR (DMSO-d6) d 169.8, 152.6, 147.5, 140.8, 138.3, 137.9,
134.7, 130.6, 129.7, 129.2, 128.6, 125.2, 124.7, 119.5, 114.7, 102.6,
44.4, 23.3; HRMS (ESI) calc. for C20H19N2O ([M - I]+) 303.1497,
found 303.1503.
2-[(E)-2-(N-Acetyl-N-phenylamino)ethenyl]quinoline methiodide
(6)
1,2-Dimethylquinolinium iodide (5, 2.85 g, 10 mmol) and N,N¢-
diphenylformamidine (3.92 g, 20 mmol) were suspended in acetic
anhydride (20 mL). The suspension was stirred at 150 ◦C for 1 h.
The reaction mixture was cooled to room temperature. Diethyl
ether (150 mL) was added to the reaction mixture with stirring.
The supernatant was discarded by decantation and the residue
was washed with diethyl ether twice. The residue was dissolved in a
small amount of dichloromethane. The solution was added to ethyl
acetate (500 mL) with stirring. The supernatant was discarded and
the residue was washed with ethyl acetate twice. Ethyl acetate was
added to the residue, and the precipitate was filtered and dried in
vacuo to give 6 as a dark green powder (3.54 g, 8.22 mmol, 82%):
1H NMR (CD3OD) d 8.98 (d, J = 13.9, 1H), 8.80 (d, J = 9.1, 1H),
8.30 (dd, J = 9.1, 0.5, 1H), 8.27 (d, J = 9.1, 1H), 8.21 (dd, J = 8.1,
1.5, 1H), 8.08 (m, 1H), 7.84 (m, 1H), 7.72–7.63 (m, 3H), 7.53–7.51
(m, 2H), 5.75 (d, J = 14.2, 1H), 4.13 (s, 3H), 2.09 (s, 3H); 13C NMR
(CD3OD) d 171.7, 158.6, 145.3, 145.1, 140.9, 139.2, 136.0, 132.0,
131.3, 129.8, 129.7, 128.9, 121.7, 119.6, 104.1, 40.0, 23.5; HRMS
(ESI) calc. for C20H19N2O ([M - I]+) 303.1497, found 303.1503.
Fluorescent dye 10
Carboxylic acid 1 (324 mg, 1.0 mmol) and 9 (430 mg, 1.0 mmol)
were suspended in dichloromethane (20 mL). Triethylamine
(1.4 mL, 10 mmol) was added to the suspension and the resultant
solution was stirred at room temperature for 16 h. The solvent
was removed by evaporation. Dichloromethane (30 mL) and a
few drops of MeOH were added to dissolve the residue and then
acetone (300 mL) was added. The precipitate was filtered, washed
with acetone and dried in vacuo to give 10 as a dark red-purple
1
solid (278 mg, 0.565 mmol, 57%): H NMR (DMSO-d6) d 8.59
(t, J = 12.8, 1H), 8.29 (t, J = 8.6, 2H), 8.06–8.04 (m, 2H), 7.85–
7.76 (m, 4H), 7.69–7.64 (m, 2H), 7.58–7.50 (m, 2H), 7.02 (t, J =
12.1, 2H), 4.35 (t, J = 7.3, 2H), 3.95 (s, 3H), 2.21 (t, J = 7.2,
2H), 1.80–1.74 (m, 2H), 1.59–1.53 (m, 2H); 13C NMR (DMSO-
d6) d 174.9, 148.3, 147.9, 142.3, 141.2, 140.3, 138.7, 137.8, 132.43,
132.37, 125.8, 125.5, 124.7, 124.5, 124.0, 123.8, 117.2, 117.1, 110.5,
110.3, 108.4, 108.2, 52.8, 41.3, 34.3, 28.2, 22.0; HRMS (ESI) calc.
for C27H27N2O2 ([M - Br]+) 411.2073, found 411.2081.
Fluorescent dye 7
Carboxylic acid 1 (324 mg, 1.0 mmol) and 6 (430 mg, 1.0 mmol)
were suspended in dichloromethane (20 mL). Triethylamine
(1.4 mL, 10 mmol) was added to the suspension and the
resultant solution was stirred at room temperature for 16 h. The
solvent was evaporated in vacuo. The residue was suspended in
dichloromethane (30 mL) and acetone (300 mL). The precipitate
was filtered, washed with acetone and dried in vacuo. The residue
was washed with dichloromethane (50 mL), filtered and dried in
vacuo to give 7 as a dark green powder (327 mg, 0.67 mmol, 67%):
1H NMR (DMSO-d6) d 8.69–8.64 (m, 1H), 8.41 (d, J = 8.3, 1H),
8.27–8.23 (m, 2H), 7.98–7.77 (m, 6H), 7.71–7.61 (m, 2H), 7.41 (t,
J = 7.2, 1H), 7.10 (d, J = 14.0, 1H), 6.47 (d, J = 13.0, 1H), 4.46 (t,
J = 7.2, 2H), 3.92 (s, 3H), 2.20 (t, J = 7.3, 2H), 1.84–1.78 (m, 2H),
1.59–1.53 (m, 2H); 13C NMR(2 : 1 DMSO-d6:CD3COOD) d 175.1,
153.2, 150.4, 145.0, 141.8, 140.7, 138.7, 135.6, 133.6, 132.8, 129.5,
126.7, 125.8, 125.2, 124.9, 120.8, 118.0, 116.7, 110.5, 109.3, 107.2,
54.1, 36.6, 33.7, 28.9, 22.3; HRMS (ESI) calc. for C27H27N2O2
([M - Br]+) 411.2073, found 411.2079.
N-Hydroxysuccinimidyl ester of fluorescent dyes
Fluorescent dye 4, 7 or 10 (80 mmol) and N-hydroxysuccinimide
(18.4 mg, 160 mmol) were suspended in DMF (2 mL). After the
addition of N,N¢-diisopropylcarbodiimide (50 mL, 160 mmol), the
reaction mixture was stirred at room temperature for 16 h.
Probe synthesis
DNA oligomers containing diamino-modified nucleotide were
prepared by a standard phosphoramidite method on a DNA
synthesizer. Diamino-modified nucleoside phosphoramidites were
synthesized according to our previous report.7 The synthesized
DNA oligomer was cleaved from the CPG support with 28%
aqueous ammonia and deprotected at 25 ◦C for 16 h. After removal
of the ammonia from the solution under reduced pressure, the
DNA was purified by reversed-phase HPLC elution with a solvent
mixture of 0.1 M triethylammonium acetate (TEAA) (pH = 7.0)
and a linear gradient over 20 min from 5 to 30% acetonitrile at a
flow rate of 3.0 mL min-1. The purified DNA was de-salted and
dissolved in distilled water. A solution of the succinimidyl ester of
the fluorescent dyes (40 mM) in DMF was added to a solution
of purified DNA in 100 mM sodium carbonate buffer (pH =
9.0) and incubated at 25 ◦C for 2 h. The reaction mixture was
diluted with ethanol. After centrifuging at 4 ◦C for 20 min, the
supernatant was removed. The residue was dissolved in a small
amount of water and then the solution was passed through a
4-[(E)-2-(N-Acetyl-N-phenylamino)ethenyl]quinoline methiodide
(9)
1,4-Dimethylquinolinium iodide (8, 2.85 g, 10 mmol) and N,N¢-
diphenylformamidine (3.92 g, 20 mmol) were suspended in acetic
anhydride (20 mL). The suspension was stirred at 150 ◦C for 1 h.
The reaction mixture was cooled to room temperature. Diethyl
ether (150 mL) was added to the reaction mixture with stirring.
The supernatant was discarded by decantation and the residue
was washed with diethyl ether twice. The residue was dissolved in a
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0.45 mm filter. The product was purified by reversed-phase HPLC
on a 5-ODS-H column, elution with a solvent mixture of 0.1 M
TEAA (pH = 7.0) and a linear gradient over 28 min from 5
to 40% acetonitrile at a flow rate of 3.0 mL min-1. After de-
salination, the probe was dissolved in a small amount of 10 mM
sodium carbonate (pH = 9.0). The fluorescent DNA was identified
by MALDI-TOF mass spectrometry (here, the molecular weight
of the counter anions of the dyes is not included in the value
of M): CGCAATD644TAACGC, calc. for C184H221N56O78P12S2
([M - H]+) 4900.9, found 4901.2; CGCAATD662TAACGC,
a humidified atmosphere with 5% CO2. For experimental use, cells
(passage numbers 5–9) were cultured in glass-based dishes. Prior
to microscope observation, the culture medium was washed and
exchanged in phenol red-free DMEM.
Transfection and cell imaging
A solution of Probe1(D715) in water (50 mM, 2 mL) was introduced
using 2 mL of LipofectamineTM 2000 (Invitrogen). After incubation
for 1 h in a solution of LipofectamineTM 2000 and probe, the
cells were washed twice with PBS and observed in phenol red-
free DMEM. Cells were maintained under culturing conditions
using an incubation system during the observation. Images were
acquired with a motorized inverted microscope (Axio Observer
Z1; Zeiss) equipped with a 20¥ objective and an XF142-2 filter
set (Optoscience, ex 685AF30, dm 708DRLP, em 730AF30). The
acquired images were analyzed and processed with AxioVision
4.2.
calc. for C188H225N56O78P12 ([M
4891.8; CGCAATD715TAACGC, calc. for C188H225N56O78P12
([M
H]+) 4888.9, found 4888.7; TTTTTTD644TTTTTT,
calc. for C188H231N34O92P12S2 ([M
H]+) 4874.9, found
-
H]+) 4888.9, found
-
-
4876.5; TTTTTTD662TTTTTT, calc. for C192H235N34O92P12 ([M -
H]+) 4862.9, found 4863.4; TTTTTTD715TTTTTT, calc. for
C192H235N34O92P12 ([M - H]+) 4862.9, found 4863.6.
Absorption and fluorescence measurements
Acknowledgements
Absorption and fluorescence spectra of the fluorescent probes
(0.4 mM) were measured in 50 mM sodium phosphate buffer (pH =
7.0) containing 100 mM sodium chloride using a cell with a 1 cm
path-length. The excitation and emission bandwidths were 1.5 nm.
We thank Dr Yayoi Hongo and Dr Takehiro Suzuki (RIKEN) for
their assistance with the mass spectrometry.
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4204 | Org. Biomol. Chem., 2011, 9, 4199–4204
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