Design and synthesis of caged fluorescent nucleotides and application to live-cell RNA imaging

Article abstract of DOI:10.1002/cbic.201100523

A binary photocontrolled nucleic acid probe that contains a nucleotide modified with one photolabile nitrobenzyl unit and two hybridization-sensitive thiazole orange units has been designed for area-specific fluorescence imaging of RNA in a cell. The synthesized probe emitted very weak fluorescence regardless of the presence of the complementary RNA, whereas it showed hybridization-sensitive fluorescence emission at 532 nm after photoirradiation at 360 or 405 nm for uncaging. Fluorescence suppression of the caged probe was attributed to a decrease in the duplex-formation ability. Caged fluorescent nucleotides with other emission wavelengths (622 and 724 nm) were also synthesized in this study; they were uncaged by 360 nm irradiation, and emitted fluorescence in the presence of the complementary RNA. Such probes were applied to area-specific RNA imaging in a cell. Only probes in the defined irradiation area were activated by uncaging irradiation, and subnuclear mRNA diffusion in a living cell was monitored.

Full text of DOI:10.1002/cbic.201100523

DOI: 10.1002/cbic.201100523
Design and Synthesis of Caged Fluorescent Nucleotides
and Application to Live-cell RNA Imaging
Shuji Ikeda,[a] Takeshi Kubota,[a] Dan Ohtan Wang,[a, b] Hiroyuki Yanagisawa,[a]
Tadashi Umemoto,[a] and Akimitsu Okamoto*[a]
A binary photocontrolled nucleic acid probe that contains a
nucleotide modified with one photolabile nitrobenzyl unit and
two hybridization-sensitive thiazole orange units has been de-
signed for area-specific fluorescence imaging of RNA in a cell.
The synthesized probe emitted very weak fluorescence regard-
less of the presence of the complementary RNA, whereas it
showed hybridization-sensitive fluorescence emission at
532 nm after photoirradiation at 360 or 405 nm for uncaging.
Fluorescence suppression of the caged probe was attributed
to a decrease in the duplex-formation ability. Caged fluores-
cent nucleotides with other emission wavelengths (622 and
724 nm) were also synthesized in this study; they were unc-
aged by 360 nm irradiation, and emitted fluorescence in the
presence of the complementary RNA. Such probes were ap-
plied to area-specific RNA imaging in a cell. Only probes in the
defined irradiation area were activated by uncaging irradiation,
and subnuclear mRNA diffusion in a living cell was monitored.
Introduction
Chemists and biologists frequently use fluorescent signals for
detecting nucleic acids and monitoring their functions, and
many fluorescent dyes have been developed for this purpose.[1]
For fluorescence monitoring of target biomolecules, drugs, or
chemicals in a cell, direct fluorescent labeling of the molecules
of interest (e.g., GFP-fused proteins) and target-specific bind-
ing of fluorescence-labeled molecules (e.g., fluorescence-la-
beled DNA) have been used. The latter labeling method is
often used for RNA imaging in a cell because direct labeling of
expressed RNA is not easy.[2] In this case, the fluorescence in-
tensity reflects the concentration of fluorescent probe but
does not always reflect the concentration of the target mole-
cule, because fluorescent dyes inherently emit fluorescence
even in the absence of their target; that is, higher intensity of
fluorescence means higher concentration of dyes, but does
not always mean higher abundance of the target molecule. It
is important to overcome this disadvantage for visualizing and
monitoring specific RNA located in a limited area in the cell of
interest. For such fluorescent imaging, fluorescent probes re-
quire 1) an increase in fluorescence by target-specific binding
and a decrease in extraneous fluorescence by nonspecific bind-
ing (to visualize only the target molecule), 2) low background
fluorescence for the suppression of fluorescence from excess
dye, and 3) high area-specific fluorescence for spatiotemporal
observation of the target molecule. The challenge of the probe
design is how to control the fluorescence of the probe. Turn-
ing off the fluorescent signal is the best way to remove back-
ground noise. A fluorophore that only emits a fluorescent
signal when it recognizes a target molecule is ideal. The
design of a fluorophore that can be area-specifically activated
by remote control, such as photoirradiation, is also desirable.
The most popular area-specific photocontrols are FRAP
(Fluorescence Recovery after Photobleaching), which monitors
the movement of unbleached fluorophores from the surround-
ing area into the bleached region after a bleaching process,[3]
and FLIP (Fluorescence Loss in Photobleaching), which moni-
tors the decrease or disappearance of fluorescence in a defined
region adjacent to a repetitively bleached region.[4] Instead of
these types of pinpoint fluorescence bleaching, the iFRAP (in-
verse FRAP) technique is occasionally used. In this area-specific
fluorescence-emission method, all the fluorescence of the
specimen is bleached except for a small region of interest, and
the postbleach images are analyzed to display the loss of fluo-
rescence from the unbleached region.[5] A molecular caging
system is effective for area-specific activation. Photolysis of a
photolabile precursor of ATP (caged ATP) is one of the well-
known examples of photoactivation of a molecular function.[6,7]
Molecular caging might be appropriate for area-specific fluo-
rescence control.
In addition to such photoirradiation-based area-specific fluo-
rescence controls, the addition of target-specific fluorescence
controls to the fluorescent probes should be a significant goal
for the design of the next generation of RNA imaging; such
probes would make possible negligible emission in the single-
stranded state (before recognition of a target RNA) and strong
[a] Dr. S. Ikeda, Dr. T. Kubota, Dr. D. O. Wang, H. Yanagisawa, Dr. T. Umemoto,
Dr. A. Okamoto
Advanced Science Institute, RIKEN
Wako, Saitama 351-0198 (Japan)
E-mail: aki-okamoto@riken.jp
[b] Dr. D. O. Wang
Institute for Integrated Cell-Material Sciences (iCeMS)
Kyoto University
Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100523.
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A. Okamoto et al.
emission upon forming a duplex
with a target RNA. Several fluo-
rescent nucleic acid probes[8,9]
have been developed for RNA
imaging in a cell. The fluores-
cence of most of them (e.g., mo-
lecular beacons)[9] is controlled
by energy transfer between two
dyes. Furthermore, to achieve
higher flexibility in probe se-
quence design and sensitive
control of fluorescence emission,
there is a need to design a fluo-
rescence-controlled probe where
both
functions—area-specific
probe initiated by photoactiva-
tion, and hybridization-sensitive
(R)-NB
Scheme 1. Structure of the caged fluorescence nucleotide
D .
514
fluorescence
emission—are
packaged into one nucleotide.
In this paper, we report a
caged hybridization-sensitive fluorescent probe for intracellular
RNA monitoring. Fluorescent probes containing a nucleotide
with a photolabile group and hybridization-sensitive fluores-
cent dyes were synthesized. They emitted very weak fluores-
cence regardless of the presence of the complementary RNA,
whereas they showed hybridization-sensitive fluorescence
emission after photoirradiation for uncaging. Such probes
were applied to live-cell RNA imaging through point photoacti-
vation, and the mobility of subnuclear RNA was monitored.
Results and Discussion
Molecular design
To provide the imaging probe with a hybridization-sensitive
fluorescent emission function, an exciton-controlled hybridiza-
tion-sensitive fluorescent oligonucleotide (ECHO) probe system
was adopted; this shows high fluorescence intensity upon hy-
bridization with its target nucleic acid, and effective quenching
of unhybridized probe.[10] The structural feature of the ECHO
probe is a pair of fluorescent dye molecules tethered to one
nucleotide in a nucleic acid probe. For the design of another
function to be added to the imaging probe, that is, a caging
and uncaging function, caged ATP gave us the solution.[6]
Caged ATP is a nucleotide analogue in which the terminal
phosphate is esterified with a blocking group, thereby render-
ing the molecule biologically inactive; ultraviolet photolysis of
the caging group results in a rapid and highly localized release
of free nucleotide at the site of irradiation.
NB
Figure 1. Uncaging and emission control of
D
-incorporated DNA probes
514
These two different photochemical systems were packaged
and the probe sequences used in this study.
NB
into one nucleotide. We designed a nucleotide,
D , that
514
contains both a hybridization-sensitive fluorescence emission
function and a photolabile blocking function (Scheme 1). The
interaction between parallel-stacked thiazole orange dyes
causes a splitting of the excited state of the dyes into two
energy levels, E’ (the out-of-phase dipole arrangement) and E“
(the in-phase dipole interaction); only the transition from the
ground state to the upper excitonic state E” is allowed,[11] and
NB
two
D
thiazole orange dyes emit fluorescence upon bind-
514
ing to the nucleic acid duplex structure, whereas before hy-
bridization these dyes show weak fluorescence because of dye
aggregation and excitonic interaction (Figure 1). The excitonic
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Caged Fluorescent Nucleic Acid Probes
Scheme 2). After the removal of camphanic acid by hydrolysis,
a methylene linker (which, together with the caging unit, is re-
moved from the nucleotide upon photolysis) was attached to
2 to give 3. The caging unit 3 was activated with sulfuryl chlo-
ride and then attached to the nucleoside with a blanched
amino linker 4, which was synthesized according to the proce-
dure previously reported[10a] under alkaline conditions to
obtain 5 (Scheme 3). Nucleoside 5 was converted into the
phosphoramidite form 6 for DNA synthesis. The phosphorami-
dite unit
(ODNs) by using a DNA autosynthesizer, and the ODN-contain-
ing was obtained after deprotection with ammonia
6 was incorporated into oligodeoxynucleotides
Figure 2. Excitonic interaction and emission control by thiazole orange dyes
in D514
.
7
(Scheme 4). The thiazole orange dye with an activated linker 8,
this rapidly deactivates to the lower excitonic state (Figure 2).
Emission from E’ is theoretically forbidden, and thus it is diffi-
cult to observe the fluorescence emission. In contrast, hybridi-
zation causes the probe to lose the excitonic interaction be-
tween the dyes through binding of each dye to the duplex
which was prepared according to an earlier report,[10a] was fi-
nally reacted with 7 in the ODN to obtain the ODN containing
NB
a caged fluorescent nucleotide,
D .
514
NB
Photolysis of
D
514
structure.
NB
Synthesized ODN1((R)-NBD514
) (Figure 1) was irradiated with
Another functional group of
D , the nitrobenzene group,
514
360 nm UV light (360ꢁ5 nm, 9.80ꢁ0.05 mWcmꢀ2). The pho-
tolysis of ODN1((R)-NBD514) was monitored by the absorbance at
260 nm on HPLC. HPLC profiles showed degradation in
ODN1((R)ꢀNBD514), with a half-life of 34 s (Figure 3). With a de-
crease in ODN1((R)-NBD514), a major signal (indicating the genera-
tion of a new product) appeared with a shorter retention time,
consistent with that of ODN1(D514), which has a structure in
which the nitrobenzene group of base N3 has been removed.
MALDI-TOF mass spectrometry identified the photolysis prod-
uct of ODN1((R)-NBD514) as ODN1(D514).
is tethered to thymine base N3, and is used for base-pairing
with the complementary adenine base. a-Methyl-2-nitro-4,5-di-
methoxybenzyl alcohol (1) was adopted for the caging unit in
this chemistry (Scheme 2). This nitrobenzyl unit has high un-
caging activity, and a 405 nm solid-state LED laser is suitable
for uncaging, as it is less harmful to living cells than UV light.
NB
The substitution at N3 prevents
D -containing DNA from
514
hybridizing with the complementary RNA, and results in sup-
NB
pression of fluorescence emission from
until uncaging.
D -containing DNA
514
Synthesis
Suppression of fluorescence emission by caging
Benzyl alcohol 1, prepared according to the procedure previ-
ously reported,[12] was coupled with (ꢀ)-camphanic chloride for
optical resolution, and the products were recrystallized into
(R)-2 (99% diastereomeric excess (de)) and (S)-2 (98% de;
ECHO probes exhibit on–off switching of fluorescence that is
dependent on addition of the complementary RNA strand.[10]
(R)-NB
However, the caged ECHO probe,
D -containing ODN,
514
emitted only weak fluorescence in the presence of the comple-
Scheme 2. Synthesis and diastereomeric resolution of a nitrobenzyl unit. a) pyridine, RT, 16 h; b) diastereomeric resolution; c) aq. KOH, RT, 6 h, 95%; d) NaH,
THF, RT, 10 min; e) NaI, CH3SCH2Cl, RT, 3 h, 54%.
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A. Okamoto et al.
Scheme 3. Synthesis of the phosphoramidite unit of a caged nucleoside. a) sulfuryl chloride, RT, 20 min; b) 4, Cs2CO3, DMF/CH2Cl2 (5:2), RT, 1 h, 22%; c) 2-cya-
noethyl N,N,N’,N’-tetraisopropyl phosphoramidite, 1H-tetrazole, acetonitrile, 258C, 1 h.
Scheme 4. Synthesis of a caged fluorescent nucleic acid. a) DNA synthesis; b) 28% aq. ammonia, 258C, 16 h; c) 100 mm sodium carbonate (pH 9.0), 258C,
10 min.
(R)-NB
(R)-NB
mentary RNA strand (Figure 4). The emission spectrum of
ODN1((R)-NBD514) (0.4 mm) excited at 513 nm showed negligible
fluorescence, and the fluorescence intensity was still low (Ff =
0.08) after addition of 13-mer adenyl RNA (A13 RNA). However,
although the solution exhibited negligible fluorescence when
irradiated at 360 nm, upon excitation at 513 nm the emission
spectrum of the solution showed a strong fluorescence band
at 532 nm when the complementary RNA was added (Ff =
0.41). The behavior of the ODN after 360 nm irradiation was
the same as that of ODN1(D514). This phenomenon was also
observed for ODN2(
D
), an
D -containing ODN with
514
514
a different sequence (Figure 1). ODN2((R)-NBD514) showed a high
fluorescence intensity at 532 nm only upon both photoirradia-
tion and addition of the complementary RNA (Ff =0.06!
0.38).
The characteristic switching of fluorescence intensity can be
explained by the absorption spectra. The absorption bands of
ODN1((R)-NBD514) were predominantly observed at 478 nm re-
gardless of the presence of complementary RNA, although a
shoulder appeared at about 510 nm (Figure 5). The absorption
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Caged Fluorescent Nucleic Acid Probes
Figure 5. Absorption spectra. Samples (0.4 mm) were measured in sodium
phosphate (50 mm, pH 7.0) and sodium chloride (100 mm) at 258C, in the
Figure 3. Conversion of ODN1((R)-NBD514) by photoirradiation. Consumption of
absence (gray line) or presence (black line) of A13 RNA. A) ODN1((R)-NBD514).
(R)-NB
(R)-NB
ODN1(
D ) and yield of the product were analyzed by HPLC. A) HPLC
514
B) ODN2(
D ).
514
profiles at each photoirradiation time. B) Changes in the amount of
(R)-NB
ODN1(
D ), (triangles) and a reaction product (circles) depend on irradia-
514
tion time. The peak area of ODN1(D514) was used as a standard for calcula-
tion of the yields of the product.
Inhibition of hybridization by caging
(R)-NB
The low fluorescence intensity of ODN1(
D ), observed in
514
the presence of the complementary RNA, arose from inefficient
hybridization. The melting temperature (Tm) of the duplex of
ODN1((R)-NBD514) and the complementary RNA was 228C (calcu-
lated from the change in the absorption at 260 nm, 10–908C).
This is much lower than that of the duplex of ODN1(D514) and
A
13 RNA (Tm =498C). ODN2((R)-NBD514) also showed lower duplex
stability with A13 RNA (Tm =188C) than that of the duplex of
ODN2(D514) and A13 RNA (Tm =548C). Addition of a nitrobenzyl
group to the ECHO probes effectively decreases the duplex-
formation ability of the probe.
Fluorescence measurement at a lower temperature induced
recovery of the hybridization-sensitive fluorescence emission.
Figure 4. Fluorescence emission spectra. Samples (0.4 mm) were measured in
sodium phosphate (50 mm, pH 7.0) and sodium chloride (100 mm) at 258C.
lex =513 nm. A) ODN1((R)-NBD514). B) ODN2((R)-NBD514).
(R)-NB
We measured the fluorescence of ODN1(
D ) at 48C, which
514
is a temperature low enough for hybridization with A13 RNA
(Figure 6). The low temperature induced recovery of the hy-
spectra suggest that the excitonic interaction remained even
when the complementary RNA was added. In contrast, the
photoirradiation product showed a hybridization-sensitive ab-
sorption shift. The product exhibited a main absorption band
at 478 nm before addition of RNA (like ODN1((R)-NBD514) before
photoirradiation), but the absorption band shifted to 509 nm
after addition of RNA, thus suggesting that the dye aggregate
was dissociated and dyes were released from their excitonic in-
teraction. Therefore, the product obtained by photoirradiation,
ODN1(D514), emitted fluorescence. The hybridization-sensitive
absorption-band shift after photoirradiation was also observed
for ODN2((R)-NBD514). Addition of a nitrobenzyl group to the
ECHO probes makes dissociation of dye aggregation ineffec-
tive.
Figure 6. Temperature effect. Samples (0.4 mm) were measured in sodium
phosphate (50 mm, pH 7.0) and sodium chloride (100 mm) at 48C (lex
=
513 nm). A) Fluorescence emission spectra. B) Absorption spectra before
irradiation, in the absence (black) or presence (gray) of A13 RNA.
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A. Okamoto et al.
bridization-sensitive fluorescence emission despite attachment
of a nitrobenzyl unit, although the fluorescence intensity in
the presence of A13 RNA was lower than that of the duplex
after photoirradiation. The photophysical behavior was like
that observed for single-base mismatched duplexes of ECHO
probes, as reported in a previous paper.[10b] An absorption shift
was observed following addition of A13 RNA at 48C, thus sug-
gesting that aggregation and dissociation of the dyes in
ODN1((R)-NBD514) were controlled, and depended on the addition
of A13 RNA. The effect of temperature on fluorescence intensity
suggests that the insensitive fluorescence response was attrib-
utable to the decrease in duplex thermostability resulting from
addition of a nitrobenzyl group.
participation of the diastereomeric effect of the nitrobenzyl
group on hybridization inhibition and photolysis was limited.
Caging and uncaging of hybridization-sensitive fluorescent
probes with different emission wavelengths
This caging system is applicable to other nucleotides with dif-
ferent fluorescence emission wavelengths. We synthesized
(R)-NB
ODNs containing caged fluorescent nucleotides,
D
in an
600
(R)-NB
orange color and
D
715
in the near-infrared, according to the
synthetic protocol for hybridization-sensitive fluorescent
probes with different emission wavelengths[10c,d] (Figure 8).
Diastereomeric isomer
The diastereomeric isomer ODN1((S)-NBD514) was also prepared,
and its photophysical properties were measured. This ODN
showed very similar fluorescence emission and absorption
spectra to those of ODN1((R)-NBD514). ODN1((S)-NBD514) showed the
characteristic fluorescence emission and absorption shifts (Fig-
ure 7). Before irradiation, small changes in fluorescence intensi-
(R)-NB
(R)-NB
Figure 8. Caging of D600 and D715. A) Structures of
B) Fluorescence emission spectra of ODN1(
D
and
D .
715
600
(R)-NB
D ) (0.4 mm, lex =596 nm) in
600
sodium phosphate (50 mm, pH 7.0) and sodium chloride (100 mm) at 258C.
(R)-NB
C) Fluorescence emission spectra of ODN1(
D ) (2 mm, lex =715 nm) in
715
sodium phosphate (50 mm, pH 7.0) and sodium chloride (100 mm) at 258C.
Their preparation was accomplished by coupling each dye by
an activated linker with (R)-7 in DNA, shown in Scheme 4. Both
ODN1((R)-NBD600) and ODN1((R)-NBD715) showed suppression of
fluorescence emission and little shift of the absorption band,
regardless of the presence of the complementary RNA. After
5 min irradiation at 360 nm, the photoreaction products from
ODN1((R)-NBD600) and ODN1((R)-NBD715) emitted fluorescence with
high intensity at 622 and 724 nm, respectively, in the presence
of complementary RNA. MALDI-TOF mass spectrometry of their
Figure 7. Absorption and fluorescence emission spectra of ODN1((S)-NBD514).
Samples (0.4 mm) were measured in 50 mm sodium phosphate (pH 7.0) and
(S)-NB
100 mm sodium chloride at 258C. lex =513 nm. A) Structure of
the emission spectra of ODN1(
D
and
514
(S)-NB
D ). B) Absorption spectra in the absence
514
(black) and presence (gray) of A13 RNA.
photolysis products indicated the production of ODN1(D600
)
and ODN1(D715), respectively. The uncaging process by photo-
irradiation was effective for caged ECHO probes with a variety
of fluorescence wavelengths.
ty and absorption band wavelength were observed in the
presence of the complementary RNA, thus suggesting that hy-
bridization inhibition by a nitrobenzyl group is effective (Tm =
208C). The large enhancement of fluorescence and the shift of
the absorption band to a longer wavelength were induced by
the addition of the complementary RNA after irradiation. The
Uncaging and fluorescence enhancement in a living cell
The caged ECHO probes were incorporated into a living HeLa
cell, which was then photoirradiated. We injected
ODN1((R)-NBD514), which binds to poly(A)+ RNA (i.e., mRNA
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Caged Fluorescent Nucleic Acid Probes
poly(A) tails), into cell nuclei by using a pneumatic injector.
The probe emitted fluorescence weakly in the cell. We irradiat-
ed the cell nucleus with blue light from a 405 nm solid-state
LED laser. After irradiation for 106 ms, the green fluorescence
in the nucleus was enhanced dramatically (Figure 9). The fluo-
Figure 9. Uncaging in a HeLa cell and increase in fluorescence. Images of an
area (128ꢁ5 pixels, white box in the DIC image) in a probe-injected HeLa
cell were acquired at high frequency (~10 ms intervals). The gray column
represents the period of laser radiation (405 nm, 376–482 ms). Bar, 10 mm.
Figure 10. Cell uncaging. A) HeLa cells. i) DIC image showing the cell nuclei
(R)-NB
where ODN1(
D ) was microinjected (cells A and C); fluorescence
514
images after ii) no laser irradiation; iii) irradiation with a 405 nm solid-state
LED laser only to cell A; and iv) irradiation with a 405 nm laser to cell C.
B) Rat nerve cells: DIC and fluorescence images showing the cell nuclei
rescence intensity increased up to 2.60-fold (ꢁ0.66, n=15) of
that of the caged state. The enhancement in fluorescence in-
tensity of ODN2((R)-NBD514) by photoirradiation (the abundance
of the intracellular target RNA is probably not so high because
the sequence is not likely to appear frequently) was limited to
(1.65ꢁ0.23)-fold (n=5) in the cell. Uncaging of the caged
ECHO probes proceeded in the cell, and the fluorescence en-
hancement reflected the abundance of probe-hybridizable
RNA in the cell.
(R)-NB
i) where ODN1(
D ) was microinjected but not photoirradiated; ii) after
514
irradiation of the right cell with a 405 nm laser. Bar, 10 mm.
and behavior of mRNA in a cell nucleus is significant for cell
biologists.[13] To detect subnuclear mRNA diffusion with a fluo-
rescence signal, the development of fluorescent probes to
bind poly(A) mRNA sequences might be one of the most effec-
tive applications. An ODN1((R)-NBD514)-injected cell initially emit-
ted weak fluorescence (Figure 11). We irradiated a small de-
fined area in a cell nucleus. Uncaging immediately occurred in
one area (area a) and showed a large enhancement of fluores-
cence. In the initial 10 s after photoirradiation, the fluorescence
started spreading out toward the surrounding area, and 20 s
later a much wider area (b and c) in the nucleus became
brighter, compared with the irradiation area. The fluorescence
of the irradiation area was initially strong but gradually re-
duced with time, whereas the fluorescence intensity of the sur-
rounding area gradually increased for 1 min after irradiation,
and then the fluorescence started to decrease. The fluores-
cence intensity of the cytoplasm area (d) was originally weak,
and no sudden change in the fluorescence intensity by photo-
irradiation was observed. However, the fluorescence increased
very slowly, thus suggesting that the RNA with which the un-
caged probe was hybridized gradually moved outside the nu-
cleus.
Area-specific fluorescence activation
Cell-specific fluorescence activation was tested with
ODN1((R)-NBD514). We prepared ODN1((R)-NBD514)-injected HeLa
cells (cells A and C in Figure 10A). Weak fluorescence was ini-
tially observed from these cells (panel ii). Laser light (405 nm)
was shone onto just one cell nucleus (cell A in panel iii); a
strong green fluorescence appeared from the irradiated cell
nucleus. Other cells remained dark. Next, when we irradiated
the dark cell nucleus (cell C in panel iv) with blue laser light,
the fluorescence recovered to almost the same level as that of
the first irradiated cell (cell A in panel iv). By the definition of ir-
radiation area, only the ECHO probe in the area was activated
by uncaging. This uncaging process was not limited to HeLa
cells. Rat nerve cells in which ODN1((R)-NBD514) had been trans-
fected by lipofection were prepared for uncaging experiments
(Figure 10B). The neuronal nuclei showed very weak fluores-
cence before irradiation (panel i). After irradiation of one soma
at 405 nm, the irradiated cell nucleus emitted fluorescence
strongly (panel ii).
Conclusions
A binary photocontrolled probe that contained a nucleotide
modified with one nitrobenzyl unit and two thiazole orange
units was designed for area-specific fluorescence imaging of
Therefore, point uncaging makes monitoring RNA diffusion
in a nucleus possible. Effective monitoring of the expression
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A. Okamoto et al.
for 16 h. After removal of ammonia from the solution under re-
duced pressure, the DNA was purified by reversed-phase HPLC,
eluted with a solvent mixture of triethylammonium acetate (TEAA,
0.1m, pH 7.0), and a linear gradient (5–45% acetonitrile, 32 min,
3.0 mLminꢀ1). The synthesized DNA was identified by MALDI-TOF
mass spectrometry: T6(R)-7T6, calcd for C149H200N31O95P12: 4317.0
[M+H]+, found: 4318.0; T6(S)-7T6, calcd for C149H200N31O95P12: 4317.0
[M+H]+, found: 4316.1; CGCAAT(R)-7TAACGC, calcd for
C145H190N53O81P12: 4343.1 [M+H]+, found: 4342.8. For determination
of the concentration of each DNA, the purified DNA was fully di-
gested with calf intestine alkaline phosphatase (50 UmLꢀ1) and P1
nuclease (50 UmLꢀ1) at 378C for 2 h. Digested solutions were ana-
lyzed by HPLC; elution was with a solvent mixture of TEAA (0.1m,
pH 7.0) and
a linear gradient (3%–10% acetonitrile, 20 min,
3.0 mLminꢀ1). The concentration was determined by comparing
peak areas with a standard solution containing dATP, dCTP, dGTP,
and dTTP (100 mm). A solution of the succinimidyl ester of thiazole
orange pentanoic acid (8)[10a] (50 equiv to an active amino group of
DNA) in N,N-dimethylformamide was added to a solution of depro-
tected DNA in sodium carbonate buffer (100 mm, pH 9.0), and in-
cubated at 258C for 10 min. The reaction mixture was diluted with
ethanol. After centrifuging (48C, 20 min), the supernatant was re-
moved. The residue was dissolved with a small amount of water,
and then the solution was passed through a 0.45 mm filter. The
product was purified by reversed-phase HPLC on a 5-ODS-H
column, and eluted with a solvent mixture of TEAA (0.1m, pH 7.0)
Figure 11. Point uncaging. A) Diffusion of the fluorescence after uncaging at
(R)-NB
an irradiation area (square) in the nucleus of an ODN1(
D
)-injected cell.
and
a
linear gradient (5–45% acetonitrile over 32 min,
514
3.0 mLminꢀ1). The concentration of the fluorescent DNA was deter-
mined as described in the DNA synthesis. The fluorescent DNA was
identified by MALDI-TOF mass spectrometry (here, the molecular
Bar=10 mm. B) Time course of the fluorescence intensities at each area (cir-
cles a–d in A)) in the cell. Labels i, ii, and iii indicate the times when the pic-
tures in A) were taken.
weight of the counteranions of dyes is not included in the value of
T6 (ODN1((R)-NBD514)), calcd for C195H240N35O97P12S2:
(R)-NB
M): T6
D
514
RNA in a cell. The probe emitted fluorescence only when it
was irradiated at 360 or 405 nm and the complementary RNA
was present. Area-specific uncaging of the probe by using a
blue laser made visualization of subnuclear RNA mobility in a
living cell possible. This system, in which the probe fluores-
cence is controlled by light of two different wavelengths,
seems to be simpler than RNA monitoring by FRAP or iFRAP,
because of the target-positive fluorescence, short photoactiva-
tion time, and pinpoint photoirradiation area.
5062.0 [MꢀH]+, found: 5062.0; T6
D
514
T6 (ODN1((S)-NBD514)), calcd
(S)-NB
for C195H240N35O97P12S2: 5062.0 [2MꢀH]+, found: 5063.3; CGCAAT(R)-
NB
D
514
TAACGC (ODN2((R)-NBD514)), calcd for C191H230N57O83P12S2: 5088.0
[2MꢀH]+, found: 5089.0; T6
D
600
T6 (ODN1((R)-NBD600)), calcd for
(R)-NB
C199H245N35O99P12: 5081.0 [2MꢀH]+, found: 5081.9; T6
D T6
715
(R)-NB
(ODN1((R)-NBD715)), calcd for C203H248N35O97P12: 5101.1 [2MꢀH]+,
found: 5101.0.
Photolysis: Photolysis of the fluorescent probes (0.4 mm) was car-
ried out in sodium phosphate (50 mm, pH 7.0) containing sodium
chloride (100 mm). The mixture was irradiated with a MAX302
xenon light source (Asahi Spectra, Tokyo, Japan) through a 360ꢁ
Experimental Section
5 nm filter (9.80ꢁ0.05 mWcmꢀ2) at 258C for 5 min. Photoproduct
General: DNA was synthesized on a H-6 DNA/RNA synthesizer
(Nihon Techno Service Co., Ushiku, Japan). Reversed-phase HPLC
was performed on Chemcobond 5-ODS-H columns (10ꢁ150 mm;
ChemcoKorea, Cheongwon-gun, Korea) on a model 305 Chromato-
graph (Gilson Inc., Middleton, WI) with a model 118 UV detector, at
260 nm. MALDI-TOF mass spectra were measured with a Reflex
spectrometer (Bruker Daltonics). UV and fluorescence spectra were
recorded on a UV-2550 spectrophotometer and RF-5300PC spectro-
fluorophotometer (Shimadzu, Kyoto, Japan), respectively.
(R)-NB
from ODN1(
D ), calcd for ODN1(D514) (C184H228N34O92P12S2):
514
4822.8 [2MꢀH]+, found: 4821.7; from ODN1((S)-NBD514), calcd for
ODN1(D514) (C184H228N34O92P12S2): 4822.8 [2MꢀH]+, found: 4821.7;
from ODN2((R)-NBD514), calcd for ODN2(D514) (C180H218N56O78P12S2):
4848.9 [2MꢀH]+, found: 4849.7; from ODN1((R)-NBD600), calcd for
ODN1(D600) (C188H232N34O94P12): 4843.6 [2MꢀH]+, found: 4845.2;
from ODN1((R)-NBD715), calcd for ODN1(D715
) (C192H235N34O92P12):
4862.9 [2MꢀH]+, found: 4863.6.
Absorption and fluorescence measurements: Absorption and
fluorescence spectra of the fluorescent probes (0.4 mm) were mea-
sured in sodium phosphate (50 mm, pH 7.0) containing sodium
chloride (100 mm) at 258C in a cell with a 1 cm path length. The
excitation and emission bandwidths were 1.5 nm.
Synthesis of 6: The synthesis of 6 (Schemes 2 and 3) from 4,5-di-
methoxy-a-methyl-2-nitrobenzyl alcohol (1) is summarized in the
Supporting Information.
Probe synthesis: DNA oligomers were synthesized by a conven-
tional phosphoramidite method in a DNA/RNA synthesizer. Com-
mercially available phosphoramidites for dATP, dGTP, dCTP, and
dTTP and synthetic phosphoramidites (R)-6 and (S)-6 were used for
DNA synthesis. The synthesized DNA oligomer was cleaved from
the support with 28% aqueous ammonia and deprotected at 258C
Melting temperature measurements: The Tm values of duplexes
(2.0 mm) were measured in sodium phosphate (50 mm, pH 7.0) con-
taining sodium chloride (100 mm). The absorbance of the samples
was monitored at 260 nm (108C–908C, heating rate: 0.58C minꢀ1).
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ChemBioChem 2011, 12, 2871 – 2880

Caged Fluorescent Nucleic Acid Probes
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Acknowledgements
We thank Dr. Yukishige Ito and Dr. Akihiro Ishiwata (RIKEN) for
specific rotation measurements, Dr. Takemichi Nakamura and Dr.
Yayoi Hongo (RIKEN) for ESI and EI mass spectrometry, and Dr.
Takehiro Suzuki (RIKEN) for the MALDI-TOF mass spectrometry.
Keywords: fluorescence spectroscopy · fluorescent probes ·
nucleotides · photochemistry · photolysis · RNA recognition
ChemBioChem 2011, 12, 2871 – 2880
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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Received: August 17, 2011
Published online on October 28, 2011
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ChemBioChem 2011, 12, 2871 – 2880