FRET-Based Mito-Specific Fluorescent Probe for Ratiometric Detection and Imaging of Endogenous Peroxynitrite: Dyad of Cy3 and Cy5

Article abstract of DOI:10.1021/jacs.6b06398

Peroxynitrite (OONO^-) is profoundly implicated in health and disease. The physiological and pathological outcome of OONO^- is related to its local concentration, and hence, a reliable OONO^- assay is highly desired. We have developed a FRET-based small-molecule fluorescent probe (PNCy3Cy5), harnessing the differential reactivity of Cy3 and Cy5 toward OONO^- by fine-tuning. It exhibits high detection sensitivity and yields a ratiometric fluorescent signal. We have exemplified that it can be applied in semiquantitative determination of OONO^- in living cells. Notably, it specifically localizes in mitochondria, where endogenous OONO^- is predominantly generated. Thus, PNCy3Cy5 is a promising molecular tool for peroxynitrite biology.

Full text of DOI:10.1021/jacs.6b06398

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Communication
A FRET-based Mito-Specific Fluorescent Probe for Ratiometric Detection
and Imaging of Endogenous Peroxynitrite: Dyad of Cy3 and Cy5
Xiaotong Jia, Qiangqiang Chen, Yingfang Yang, Yao Tang,
Rui Wang, Yufang Xu, Weiping Zhu, and Xuhong Qian
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 12 Aug 2016
Downloaded from http://pubs.acs.org on August 12, 2016
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A FRET-based Mito-Specific Fluorescent Probe for Ratiometric De-
tection and Imaging of Endogenous Peroxynitrite: Dyad of Cy3 and
Cy5
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Xiaotong Jia†, Qiangqiang Chen‡, Yingfang Yang‡, Yao Tang†, Rui Wang‡, Yufang Xu†, Weiping
Zhu†, Xuhong Qian*†
†State Key laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, ‡Shanghai Key La-
boratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Meilong Road
130, Shanghai 200237, China
Supporting Information Placeholder
ABSTRACT: Peroxynitrite (OONO-) is profoundly implicat-
allowing self-calibration. Recently, we and other groups have
reported a few reaction-based8 small-molecule and semicon-
ed in health and disease. The physiological and pathological
ducting polymer probes for ratiometric detecting OONO-. 9-11
outcome of OONO- is related to its local concentration and
hence a reliable OONO- assay is highly desired. We have
Nevertheless, it remains challenging to design robust assays
for determining OONO- levels in living cells.
developed a FRET-based small-molecule fluorescent probe
(PNcy3cy5), harnessing the differential reactivity of Cy3 and
Cy5 toward OONO- by fine-tuning. It exhibits high detection
sensitivity and yields a ratiometric fluorescent signal. We
have exemplified that it can be applied in semi-quantitative
determination of OONO- in living cells. Notably, it specifical-
ly localizes in mitochondria, where endogenous OONO- is
predominantly generated. Thus, PNCy3cy5 is a promising
molecular tool for peroxynitrite biology.
We present herein the first FRET-based small-molecule ra-
tiometric fluorescent probe (PNcy3cy5) for detection of
OONO-. It specifically localizes at mitochondria, the major
sources of endogenous OONO-. It also exhibits high detec-
tion sensitivity and applications for semi-quantitative detec-
tion of OONO- in living cells.
Scheme 1. Structures of PNcy3cy5 and its detection
mechanism.
Peroxynitrite (OONO-) is endogenously produced in living
systems by the diffusion-controlled coupling of nitric oxide
-
(•NO) and superoxide radical anion (O2 •).1 Though the cyto-
toxicity of OONO- plays a key role in signal transduction and
anti-microbial activities,2 excessive OONO- can damage criti-
cal cell components, including proteins, DNA, lipids, iron-
sulfur clusters and thiols. The accumulation of cell injuries
eventually leads to apoptosis and necrosis.3 Mounting evi-
dences have established a firm correlation between OONO-
and a number of pathological conditions, such as cardiovas-
cular diseases, neurodegenerative diseases, chronic inflam-
mation, ischemia reperfusion injury and diabetes.4 Therefore,
assays for OONO- detection are important for elucidating
pathophysiology of OONO- and may be used for peroxyni-
trite-related disease diagnosis.
The ratiometric detection mechanism of PNcy3cy5 is
based on modulating FRET between Cy3 and Cy5. The photo-
fading and photoswiching of cyanine dyes are caused mainly
by oxidants and nucleophiles, through cleaving or forming
adducts of polymethine bridge.12 The chemostability of a
polymethine cyanine dye toward oxidants deteriorates as its
conjugative backbone elongates.12 Nagano et al. reported that
Cy5 and Cy7 are both readily oxidized by OONO- and Cy7 is
more susceptible than Cy5.5a We envisioned that Cy3, which
has shorter polymethine chain, may show better stability to
OONO-. The reactivity of Cy3 and Cy5 to OONO- was tested
in physiological pH. In contrast to Cy5, the UV-Vis absorp-
tion of Cy3 remained unchanged even with addition of 50
equiv. of OONO-, which is consistent with our hypothesis
(Figure S2). This result suggests the possibility to create a
Recently, there have emerged a number of fluorescent
probes for OONO-,5 and some of them have been used for
biomedical studies.6 Most of these probes exhibit a turn-on
fluorescence signal at a single channel. The scope of such
probes in quantitative in vitro studies is limited due to com-
plications by photo-bleaching, uneven loading, or fluctuation
in excitation intensity.7 In comparison, probes based on two-
channel ratiometric signal address these complications by
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OONO- probe by fine-tuning reactivity of Cy3 and Cy5. Also,
(Figure 1c, inset). The detection mechanism of PNcy3cy5
toward OONO- was in agreement with literature prece-
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Cy3 and Cy5 are a popular pair of FRET fluorophores. There-
fore, it is possible to design covalent Cy3-Cy5 to achieve a
FRET-based ratiometric probe for OONO-. The judicious
choice of linker between Cy3 and Cy5 is also a key factor de-
termining the energy transfer efficiency. Moerner et al. and
dents.12c The structure of the detection product (PNcy3) was
further unambiguously confirmed by MALDI-TOF ([M]+
=
m/z 740.4025).
Reactivity of PNcy3cy5 toward various potentially interfer-
ing species, including ROSs and physiological nucleophiles,
were tested (Figure 1d, Figure S11). ClO- up to 5 equiv. in-
duced a minor signal enhancement of 28-fold, while OONO-
triggered a remarkable enhancement of emission ratio
Squier
et
al.
have
employed
the
hexanoyl-
hydrazine/ethylenediamine-hexanoyl spacer to tether Cy3-
Cy5.13 The latter linker is long at ca. 21 Å. We chose a shorter
acetyl-piperazyl-hexanoyl linker of ca. 14 Å in hope for en-
hancing FRET efficiency, as which is inversely proportional
to the sixth power of the distance between the FRET pair.14
9
F560/F660 of over 300-fold as we have noted. Spectral changes
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induced by addition of other various ROSs up to 100 equiv.,
-
including H2O2, O2 •, •OH, 1O2 and ROO•, are not discernible.
-
Other physiological nucleophiles, such as HSO3 , SO32-, H2S,
Cys, Hcy and GSH also induced no obvious changes.
Figure 2. Confocal fluorescence images for intracellular lo-
calization of PNcy3cy5 in macrophages. Cells were treated
with 10 μM PNcy3cy5 for 0.5 h and stained (0.5 h) with 200
nM Mito-Tracker Green or 200 nM Lyso-Tracker DND-26 or
5 μg/mL Hoechst 33342. Red channel at 640-680 nm. Green
channel at 500-540 nm for Mito-Tracker Green and Lyso-
Tracker Green， 440-480 nm for Hoechst 33342.
Figure 1. (a) UV-Vis absorption spectra of PNcy3cy5 (2 μM)
to OONO- (0-5 equiv.); (b) Fluorescence spectra of PNcy3cy5
(2 μM) to OONO- (0-5 equiv.), inset shows the plot of emis-
sion ratio (F560/F660) to OONO- (0-10 μM), λex = 530 nm; (c)
Linear relationship of emission intensity ratio (F560/F660) to
OONO- (0-700 nM), inset shows the linear response to lower
OONO- (0-12 nM); (d) Emission intensity ratio (F560/F660) of
PNcy3cy5 (2 μM) in the presence of various ROS and some
physiological nucleophiles (5 equiv. for ClO- and OONO-, 100
equiv. for other species). Data were collected at 25 oC in 0.1 M
phosphate buffer (pH = 7.4, 0.2% DMF, v/v) within 30s.
The co-localization experiments of PNcy3cy5 were per-
formed in RAW264.7 by co-staining with commercially avail-
able Mito-Tracker Green, Lyso-Tracker Green and Hoechst
33342 (a nucleus-specific dye) (Figure 2). The red-channel
fluorescence of PNcy3cy5 showed excellent overlay with
green-channel fluorescence of Mito-Tracker Green, with
Pearson’s correlation coefficient at 0.94 ± 0.04 (evaluated by
Image J software), probably due to the ammonium cation of
cyanine fluorophore.5d, 15 Comparatively, no good overlap was
observed between PNcy3cy5 and Lyso-Tracker Green or
Hoechst 33342. In addition, the low cytotoxicity of PNcy3cy5
was also demonstrated by MTT assay (Figure S1).
The spectral properties of PNcy3cy5 were first examined in
0.1M phosphate buffer (pH = 7.4, 0.2% DMF, v/v). It exhibits
two absorption maxima at 540 nm and at 640 nm, which
respectively correspond to that of the Cy3 and Cy5 unit in its
scaffold. When PNcy3cy5 was excited at 530 nm, which effi-
ciently excites Cy3 but not Cy5, an intense fluorescence emis-
sion of Cy5 at 660 nm was observed, but not that of Cy3 at
560 nm. This indicates efficient energy transfer between Cy3
donor and Cy5 acceptor (Figure S8). Upon addition of
OONO- (0-5 equiv.), the absorption intensity of PNcy3cy5 at
640 nm decreased, while the absorption band at 540 nm re-
mained unaffected (Figure 1a). Concomitantly, the color of
the solution changes from dark purple to pink. Fluorescence
titration of a solution of PNcy3cy5 with OONO- demonstrat-
ed a decrease of the emission band at 660 nm and increase at
560 nm (Figure 1b). The fluorescence intensity ratio between
560 nm and 660 nm (F560/F660) exhibited a nearly 324-fold
enhancement, from 0.04 in the absence of OONO- to 12.96
upon addition of OONO- (5 equiv.) (Figure 1d). The emission
intensity ratio has an excellent linear relationship with
OONO- in 0-700 nM (Figure 1c). And the detection limit of
PNcy3cy5 for OONO- was tested to be as low as 0.65 nM
The ability of PNcy3cy5 for ratiometric fluorescence detec-
tion of exogenous OONO- in living cells was examined.
RAW264.7 loaded with PNcy3cy5 alone showed negligible
fluorescence in green channel and strong fluorescence in red
channel (Figure 3A, 3G). A dramatic drop of the red channel
fluorescence and remarkable enhancement in the green
channel fluorescence were observed in cells treated with SIN-
1, a OONO- donor (Figure 3B, 3C). The maximal production
rate of OONO- from SIN-1 was reported to be ca. 1.4% of ini-
tial SIN-1 concentration.16 However, cells treated with NOC-
18 (Diethylenetriamine), a NO• donor (Figure 3D, 3G) and
-
MSB (menadione sodium bisulfite), a O2 • donor (Figure 3E,
3G) were almost unaffected. In SIN-1 treated cells, a 4.8-fold
and a 29-fold and enhancement in the fluorescence ratio
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were observed (Figure 3G). We also found that the enhance-
capable of detecting the endogenous OONO- in living sys-
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ment in the fluorescence ratio (green/red channel) shows a
linear relationship with the dose of SIN-1, suggesting that
PNcy3cy5 is potentially suitable for semi-quantitative studies
(Figure 3H). Furthermore, the enhancement in emission ra-
tio induced by SIN-1 was significantly abated by the presence
of minocycline, a OONO- scavenger (Figure 3F, 3G). These
results suggest that PNcy3cy5 can specifically and semi-
quantitatively ratiometric detect and image exogenous
OONO- in living cells.
tems.
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Figure 4. Confocal fluorescence imaging of endogenous
OONO- with PNcy3cy5 in stimulated RAW264.7 macrophag-
es. Normal cells (A) were treated with only 10 μM PNcy3cy5
for 0.5 h. Stimulated cells (B, C, D) were treated with 1 μg/mL
LPS, 50 ng/mL γ-IFN and various inhibitors for 4h, then 10
μM PNcy3cy5 for 0.5 h, following 20 nM PMA for 0.5 h. (A)
control; (B) LPS/γ-IFN; (C) LPS/γ-IFN and 100 nM 1400W; (D)
LPS/γ-IFN and 100 μM apocynin. Green Channel at 540-580
nm; red channel at 640-680 nm. Ratio images generated
from green/red channel. (E) Average intensity ratios from
ratio images of A-D. Error bars represent standard deviation.
It is known that mitochondria are the primary sources of
peroxynitrite. OONO- could induce mitochondria damages
by oxidation, nitration and nitrosation of mitochondrial
components.18 The performance of PNcy3cy5 for endogenous
OONO- in mitochondria was established. RAW264.7 treated
with free PNcy3cy5 showed weak green fluorescence and
intense red fluorescence (Figure 5A). In RAW264.7 stimulat-
ed with LPS/IFN-γ for 4h and then PNcy3cy5 and PMA, it
was observed that red emission diminished and green emis-
sion increased significantly, along with outstanding overlay
with blue fluorescence from Mito-Tracker Green. To the best
of our knowledge, it has not been reported previously a
small-molecule probe for ratiometric imaging of endogenous
OONO- in mitochondria.
Figure 3. Confocal fluorescence imaging of PNcy3cy5 selec-
tivity with various exogenous ROS donors in RAW264.7 mac-
rophages. Cells were treated with 10 μM PNcy3cy5 for 0.5 h
and then corresponding stimulus for 2 h. (A) control; (B) 100
μM SIN-1; (C) 1 mM SIN-1; (D) 500 μM NOC-18; (E) 100 μM
MSB; (F) 1 mM SIN-1 and 100 μM minocycline. Green Chan-
nel at 540-580 nm; red channel at 640-680 nm. Ratio images
generated from green/red channel. (G) Average intensity
ratios from ratio images of A-F. (H) Linear relationship of
average intensity ratio with SIN-1 concentration. Error bars
represent standard deviation.
Next, the potentials of PNcy3cy5 in detection of endoge-
nously generated OONO- was evaluated in RAW264.7 mac-
rophages, which is known to release OONO- upon stimula-
tion of lipopolysaccharide (LPS)/interferon-γ (IFN-γ) and
phorbol 12-myristate 13-acetate (PMA).17 It is assumed that
the formation of OONO- in stimulated macrophages can be
regulated by nitric oxide synthase (iNOS) and NADPH oxi-
dase (NOX).17 In RAW264.7 treated with only PNcy3cy5, a
slight green emission and strong red emission were observed
(Figure 4A, 4E). Upon incubation with LPS/IFN-γ for 4h and
then PNcy3cy5 and PMA, RAW264.7 showed a marked de-
crease in red channel and a dramatic increase in green chan-
nel, resulting in a 22-fold enhancement of emission ratio
(Figure 4B, 4E). With the equation in Figure 3H, the level of
endogenous OONO- from stimulated cells was equivalent to
that from ca. 760μM SIN-1 treated cells. As expected, cells
pretreated with iNOS inhibitor (1400W) (Figure 4C, 4E) or
NOX inhibitor (apocynin) (Figure 4D, 4E) accompanied by
LPS/IFN-γ stimulation, and then PNcy3cy5 and PMA, did not
trigger the distinguishable increase of emission ratio. These
results suggest that PNcy3cy5 is a sensitive fluorescent probe
Figure 5. Confocal fluorescence imaging of endogenous
OONO- with PNcy3cy5 at mitochondria in stimulated
RAW264.7 macrophages. Cells were treated with 1 μg/mL
LPS, 50 ng/mL γ-IFN for 4h, 10 μM PNcy3cy5 for 0.5 h, 20 nM
PMA for 0.5 h, and then stained with 200 nM Mito-Tracker
Green for 0.5 h. (A) control; (B) LPS/γ-IFN and PMA. Green
Channel at 540-580 nm; red channel at 640-680 nm; ratio
images generated from green/red channel.
In conclusion, we have developed a FRET-based fine-
tuning reactivity probe (PNcy3cy5) for ratiometric detection
of peroxynitrite. In the presence of OONO-, a fluorescence
intensity increase at 560 nm and a decrease at 660 nm were
observed. The emission ratio (F560/F660) displays a large dy-
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namic increase of up to 324-fold. An excellent detection limit
of 0.65 nM was measured. PNcy3cy5 is feasible for cellular
imaging studies. It freely permeates cell membrane and spe-
cifically localizes in mitochondria, the major source of en-
dogenous peroxynitrite. PNcy3cy5 can be used in semi-
quantification of cellular OONO-, because the emission ratio
of the green channel (540-580 nm) and red channel (640-680
nm) is linear to the concentration of added SIN-1. With
PNCy3cy5, the endogenous OONO- generated in stimulated
macrophage cells was successfully detected. These results
make it a promising candidate for elucidating the involve-
ment of OONO- in various biological processes in living cells.
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ASSOCIATED CONTENT
Supporting Information
Experimental details for chemical synthesis of all compounds,
supplementary absorption and fluorescent characterization
of probe and intermediates to ROSs, and imaging methods
and data. This material is available free of charge via the In-
ternet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
xhqian@ecust.edu.cn
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by the financial support from Na-
tional Natural Science Foundation of China (21236002,
21476077, 21302125), and the Fundamental Research Funds
for the Central Universities. We thank Prof. Youjun Yang for
insightful discussions and help with manuscript preparation.
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ACS Paragon Plus Environment