A FRET-based probe with a chemically deactivatable quencher

Article abstract of DOI:10.1039/c2cc17542h

A new concept of a chemically deactivatable quencher is proposed for a FRET-based probe that turns-on its fluorescence by either an enzymatic cleavage or a chemical reagent (sodium dithionite). This concept allowed us to quantify the caspase-3 cleavage activity in solution and to reveal unreacted probes in cell experiments. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc17542h

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A FRET-based probe with a chemically deactivatable quencherw
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Geoffray Leriche, Ghyslain Budin, Zeinab Darwich, Denis Weltin, Yves Mely,
Andrey S. Klymchenkob and Alain Wagner*a
Received 2nd December 2011, Accepted 27th January 2012
DOI: 10.1039/c2cc17542h
A new concept of a chemically deactivatable quencher is proposed
for a FRET-based probe that turns-on its fluorescence by either an
enzymatic cleavage or a chemical reagent (sodium dithionite). This
concept allowed us to quantify the caspase-3 cleavage activity in
solution and to reveal unreacted probes in cell experiments.
is particularly important for intracellular studies where the probe
localisation and concentration are difficult to predict.
We selected an azo-based molecule as a suitable quencher5 and
dithionite as a chemical reagent of relatively low toxicity.6 The azo-
groups are known to react readily and under mild reduction
conditions with dithionite7 leading to NQN bond cleavage. Con-
sequently, when the azo-quencher is linked to a fluorescent dye,
dithionite can deactivate the quencher and turn-on the fluorescence.
Fluorescence is widely used to study biological processes in living
cells. Recently, turn-on probes have been developed to measure
cell enzymatic activity, pH alterations and to detect singlet
oxygen, nitric oxide and highly reactive oxygen species (hROS).1
Common turn-on probes are designed by linking a fluorescent
dye and a quencher together via a biologically labile covalent bond.
The most frequently used quencher moieties are non-emitting dyes,
which act as FRET acceptors for the covalently bound fluorescent
dye. The fluorescence is often turned-on by the enzymatic cleavage
of the linker, resulting in separation of the fluorescent dye from the
quencher (Scheme 1A). This strategy was used to image different
enzymes activities such as cathepsins, caspases and MMPs.2
Recently, this approach was also used to image free thiols in cells
by the cleavage of either a disulfide bridge3 or a vinyl sulfide group.4
Currently used turn-on fluorescent probes are not detectable
until activation leading to a high signal-to-noise ratio, which is
advantageous for live cell imaging. However, in the case where
there is no or weak signal, it is impossible to assess whether this is
due to weak biological activity, insufficient probe concentration
or inappropriate cellular localization of the probe. Having the
ability to detect the substrate entering within the cells would have
far reaching applications in biology.
Dabcyl (4-(40-dimethylamino-phenylazo)benzoic acid) is
a
common azo-based quencher widely used in a variety of biomole-
cular applications and nucleic acid probes.8 To the best of our
knowledge no information on Dabcyl reduction with dithionite has
been published. The Dabcyl dye was then incubated with a 1 mM
sodium dithionite solution in phosphate buffer (pH 7.4, 100 mM)
and the azo bond cleavage was monitored using UV spectro-
photometry at 460 nm. The half-life of Dabcyl was longer than
13 minutes (Fig. S1, ESIw) and could not be transposed to live
cells. To overcome this low reactivity, we used a highly reactive
(4-hydroxy-2-methoxy-phenylazo) benzoic acid CDQ (Fig. 1) as a
suitable chemically deactivatable quencher candidate.9 This mole-
cule has a UV profile similar to Dabcyl, but undergoes a faster
cleavage. With a 1 mM sodium dithionite solution, this compound
exhibited a half-life of less than 1 second and total cleavage was
achieved in less than 15 seconds (Fig. S1 and S2, ESIw).
Considering these results, we linked the chemically deactivatable
quencher (CDQ) to a fluorescent dye. Its absorption spectrum
overlaps with the emission of the 7-diethylaminocoumarin-3-
carboxylic acid (DEAC), which has a 470-nm emission maximum
(Fig. S3, ESIw). The corresponding chemically deactivatable
probe 2 (Fig. 1) was synthesized in five steps with an overall
yield of 19% (see ESIw).
Quencher deactivation kinetics was investigated by adding
dithionite (1 mM) to the FRET-based probe 2. A rapid 17-fold
increase of fluorescence intensity was observed following cleavage
of 2, with a short half life of B10 seconds (Fig. 2A and B). We
verified the photostability of the fluorescent dye 1 and its stability
towards dithionite (Fig. 2A). The deactivation of 2 in the presence
of dithionite was complete, as it can be seen from the disappear-
ance of its absorption peak at 490 nm (Fig. S4, ESIw). Since 2
showed very good quencher deactivation properties, we used
this couple of CDQ/DEAC groups in the design of a fluoro-
metric dual turn-on caspase-3 sensitive probe.10
The strategy developed here relies on the design of a probe
whose fluorescence can be turned-on either by a biological stimulus
or by a chemical reagent. The probe contains a central biosensitive
bond that links a fluorescent dye and a chemically deactivatable
quencher. When the labile bond is cleaved, the fluorescence is
turned-on enabling the biological stimulus to be detected
(Scheme 1A). Then, a chemical reagent capable of deactivating
the quencher can be used to turn-on fluorescence of the unreacted
probe (Scheme 1B), allowing estimation of biological cleavage. This
a Laboratory of Functional Chemo-Systems UMR 7199, 74 Route du
Rhin, 67401, Illkirch, France. E-mail: wagner@bioorga.u-strasbg.fr;
Fax: +33 368854306
b Laboratoire de Pharmacologie et Physicochimie, UMR 7213 CNRS,
74 Route du Rhin, 67401, Illkirch, France
´
We synthesized a dual probe: CDQ-DEVD-G-DEAC 3.
This probe consists of three parts: an L-amino acid effector
caspase-3 recognition sequence (DEVD)11 that is flanked by
c
PhytoDia SAS, Boulevard Sebastien Brant, 67412 Illkirch, France
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc17542h
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Scheme 1 Principle of FRET-based probe with a chemically deactivatable quencher. (A) In the presence of biological stimuli, the labile bond is cleaved
and the fluorescence is turned-on enabling the biological stimuli to be detected. (B) The introduction of a chemically deactivatable quencher allows us to
reveal the presence of an inactivated probe by treatment with a chemical reagent.
Fig. 1 Structures of cleavable quenchers (Dabcyl and CDQ),
modified DEAC 1, and probe 2.
Fig. 3 Response to caspase-3 cleavage and dithionite quencher
deactivation. (A) Cleavage kinetics of 3 at 2 mM by human recombinant
caspase-3 at 25 or 0 ng mLꢀ1 (black and red lines, respectively). The
arrow indicates the addition of dithionite (1 mM for 1 min) to cleave
probe excess. (B) Conversion rates for 3 and 4 calculated from dithionite
cleavage or AMC standard, respectively.
observed, dithionite (1 mM, 1 min) was added to the reaction
mixture with 3. A 3.1-fold fluorescence increase was observed,
indicating that some unreacted probe was still present. This
incomplete conversion might be imputed to enzyme inhibition by
the reaction product. The chemical deactivation of the quencher
enables us to calculate the total concentration and the conversion
rate of the fluorescent substrate (Fig. 3B). After 300 minutes, 30%
of 3 had been enzymatically cleaved by the recombinant caspase-3.
To estimate the amount of uncleaved probe 4, the fluorescence
intensity was compared to the free fluorescent dye, AMC, at the
same concentration (2 mM). A 3.5-fold difference was recorded,
which is similar to the signal observed for probe 3 (Fig. S6, ESIw).
Thus, the chemically deactivatable quencher allows direct
estimation of the probe conversion rate.
Fig. 2 Quencher deactivation analysis in FRET-based probe. (A)
Deactivation kinetics of 1 (dashed blue line) and 2 (dashed red line) with
1 mM of sodium dithionite. Photo-stability of 1 (solid blue line) and 2 (solid
red line). Excitation and emission wavelengths were 430 and 476 nm,
respectively. (B) Emission fluorescence spectra of compounds 1 (blue lines)
and 2 (red lines) before (solid lines) and after deactivation with 1 mM of
dithionite solution (dashed lines). All data were recorded with a probe
concentration of 2 mM in phosphate buffer (pH 7.4, 100 mM). (C) Cuvettes
under a UV-lamp containing 2 (20 mM) without and with dithionite (1 mM).
the chemically deactivatable quencher (CDQ) and the fluorescent
dye (DEAC). Probe 3 was synthesized in 12 steps using Fmoc
chemistry in solution (see ESIw). After the quencher moiety of 3
was deactivated by dithionite, the fluorescence signal was increased
by 22-fold (Fig. S5, ESIw). Thus, the quenching efficacy and
therefore the turn-on property of probe 3 was as good as those
of probe 2, despite the longer peptide linker length in the former.
In vitro enzyme kinetic studies were performed using human
recombinant caspase-3 enzyme, probe 3, and the commercially
available turn-on fluorescent substrate Ac-DEVD-AMC 4,
used as control (AMC = 7-amino-4-methylcoumarin). Both
probes were incubated with caspase-3 at 25 1C in buffer and
showed comparable fluorescence enhancement kinetics
(Fig. 3A and Fig. S6, ESIw). This result indicates that the
CDQ/DEAC pair does not perturb the enzymatic activity of
caspase-3. After 300 minutes, when saturation curves were
In the control experiment, a competitive inhibition study of
caspase-3 was performed using a commercially available inhibitor,
Ac-DEVD-CHO 5 (Fig. S7, ESIw). As expected, an increase in the
inhibitor concentration slowed down the enzymatic cleavage.
The chemical activation of 3 was then studied in tissue
culture. HeLa cells were incubated for 1 hour with 3. After
washing out probe excess, cells were incubated with dithionite.
In the control experiment, cells were treated with HEPES buffer,
instead of dithionite (Fig. 4A). The quencher deactivation was
monitored using live-cell imaging (Fig. 4). Fluorescence could be
clearly seen in dithionite treated cells after 15 minutes and the
intensity continued to increase for 45 minutes. After 45 minutes, a
maximum intensity was reached indicating that all the probes were
chemically turned-on. It confirms that dithionite can abolish the
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(Fig. 4G and G0). In contrast, cells with a healthy morphology had
only very low levels of fluorescence (Fig. 4H and H0), in line with
data on non-treated HeLa cells (Fig. 4A). The quencher was
further deactivated in the cells by 10 mM dithionite treatment for
45 minutes. A strong increase in fluorescence was observed in both
apoptotic and healthy cells (Fig. 4I and I0), indicating the presence
of uncleaved probe 3 in the both cell populations and confirms the
presence of the probe in non-fluorescent healthy cells. Thus, we
have shown that the fluorescence of probe 3 is only turned-on
in apoptotic cells, and the ‘‘chemically deactivatable quencher’’
confirms the presence of the probe in all cells.
In conclusion, we have developed a caspase-3 activity-sensitive
probe containing a DEVD peptide which was used to link a
fluorescent dye and a ‘‘chemically deactivatable quencher’’. The
synthesized probe was shown to turn-on its fluorescence in
response to both recombinant and endogenous caspase-3 stimuli,
whereas it can be turned-on independently by dithionite deactiva-
tion of the quencher. The advantages of this novel strategy over
traditional ‘‘turn-on’’ probes include control for internalization
and localization of the probe in living cells, irrespective of whether
they contain the biological stimulus or not. The ‘‘chemically
deactivatable quencher’’ concept can be applied to all ‘‘turn-
on probes’’ with a fluorescent dye-linker-quencher structure
for assaying different biological stimuli. These results pave the
way for the development of a new generation of enzymatic
probes containing an internal control.
Fig. 4 Fluorescence and brightfield microscopy images of HeLa cells
treated with 3 (40 mM, 1 h). (A–F) Quencher deactivation kinetics with
dithionite (10 mM) at different time points: 15 (B), 30 (C), 45 (D), 60 min
(E). In the control experiment (A), the cells were incubated with HEPES
buffer. (F) Brightfield image of cells after 60 min of dithionite treatment.
(G–H0) Response of 3 to apoptotic cells (pre-treated with Actinomycin D
(0.5 mg mLꢀ1, 18 h)). (I and I0) Quencher deactivation with dithionite
(10 mM, 45 min) in apoptotic cells. The images were acquired with the same
camera settings, but with a different fluorescence scale ranging from
2700 to 7360 (G–H) and from 7880 to 36 010 (all others). The image size
was 219 ꢁ 163 mm (A–H0) and 146 ꢁ 109 mm (I and I0).
We thank D. Dujardin and R. Vauchelles from PIQ imaging
platform for help with fluorescence imaging.
Notes and references
1 H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke and Y. Urano,
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FRET-quenching in 3 in healthy cells. Compared to an in vitro
experiment, in vivo dithionite-mediated quencher deactivation is
much slower due to the low dithionite membrane permeability. It
should be noted that the cells treated with dithionite showed slightly
different morphology, which could be connected with cell shrinkage
(Fig. S8, ESIw). However, after dithionite treatment the morpho-
logy remained unchanged for the experiment timescale. According
to luciferase ATP assay, the dithionite cytotoxicity was low, even at
a 10 mM concentration (Fig. S9, ESIw). Numerous reports showed
that dithionite can be used as a reductive agent in cell culture,
without strong cytotoxicity effects at the millimolar range.12
Then we assessed cell internalization of probe 3 as a function
of incubation time. Cells were treated with 40 mM of 3 for
different times (15, 30, 60 and 120 min), washed and incubated
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hour of incubation, and then reached a plateau. This allowed us
to determine the optimal incubation time of the cells with the
probe, which would not be possible for classical turn-on probes.
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cleaved in these cells. In addition, we observed different levels of
fluorescence intensity for the Actinomycin-D-treated cells, which is
most likely due to the cells being in different stages of apoptosis
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This journal is The Royal Society of Chemistry 2012