A red-emissive and positively charged RNA ligand enables visualization of mitochondrial depolarization and cell damage

Article abstract of DOI:10.1016/j.saa.2021.119686

In this work, a red-emissive RNA ligand bearing two positive charges were developed for the visualization of mitochondrial depolarization, via the subcellular localization of the ligand molecules. The ligand with quinolinium moiety and strong electronic d

Full text of DOI:10.1016/j.saa.2021.119686

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119686
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A red-emissive and positively charged RNA ligand enables visualization
of mitochondrial depolarization and cell damage
Dingyi Guo a, Jie Sun a, Minggang Tian a, Weiying Lin a,b,
⇑
a Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan,
Shandong 250022, PR China
b Guangxi Key Laboratory of Electrochemical Energy Materials, Institute of Optical Materials and Chemical Biology, School of Chemistry and Chemical Engineering, Guangxi
University, Nanning, Guangxi 530004, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
A cationic red-emissive RNA ligand is
constructed to detect mitochondrial
depolarization.
The probe targets mitochondria in
live cells and migrates into nucleolus
after loss of
DWm.
Two cations are decorated on the
probe which enables fast response to
D
Wm.
Oxidative damage of live cells is
clearly observed via subcellular
immigration of the probe.
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, a red-emissive RNA ligand bearing two positive charges were developed for the visualization
of mitochondrial depolarization, via the subcellular localization of the ligand molecules. The ligand with
quinolinium moiety and strong electronic donor displays red fluorescence peaked at 630 nm. Meanwhile,
the probe is concentrated in mitochondria of live cells due to the high mitochondrial membrane poten-
tial, and re-localizes into nucleolus upon mitochondrial depolarization owing to the affinity to RNA. In
this manner, the decrease of mitochondrial membrane potential could be real-timely and in-situ moni-
tored with the red-emissive probe. Particularly, two cations were decorated on the probe, which enables
the fast response to mitochondrial depolarization with elevated sensitivity. Cell damage induced by H2O2
was also successfully observed with the probe. We expect that the probe can promote researches on
mitochondrial membrane potential, cell apoptosis, and relative areas.
Received 25 November 2020
Received in revised form 24 February 2021
Accepted 4 March 2021
Available online 10 March 2021
Keywords:
Fluorescent probe
Mitochondrial depolarization
RNA ligand
Mitochondria
Ó 2021 Elsevier B.V. All rights reserved.
1. Introduction
mitochondria [4]. Therefore, a negatively inside transmembrane
potential up to ꢁ180 mV is formed in mitochondria, namely the
Mitochondrion is a crucial organelle in eukaryotic cells, which
plays indispensable roles in providing intracellular energy, sig-
nalling, and many other biological events [1–3]. Particularly, aero-
bic respiration process occurs in mitochondrial inner membrane
and matrix, during which two net protons were pumped out of
mitochondrial membrane potential (DWm) [5,6]. DWm plays signif-
icant roles in the generation of ATP, signaling, and energy transi-
tion. Depolarization of Wm is an important sign of cell
D
apoptosis, and also crucial for the signaling of autophagy process
to control the quality of mitochondria in live cells [7–9]. Abnormal
regulation of
DWm is closely related to Alzheimer’s disease, dia-
betes, cancer, and many other diseases [10–13]. Therefore, moni-
⇑
Corresponding author.
E-mail address: weiyinglin2013@163.com (W. Lin).
https://doi.org/10.1016/j.saa.2021.119686
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.

D. Guo, J. Sun, M. Tian et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119686
toring
fundamental significance for biology and pathology.
Determination of Wm had been a tough task in the past,
because mitochondria exhibit small size (generally 1–3 m) that
is hard to insert an electrode [14]. The Wm-dependent accumula-
tion of membrane-permeable cations into mitochondria provided a
valid way for assessment of Wm [15,16]. Fluorescent organic
cations were utilized to monitor Wm, in light of the superior
advantages of fluorescence imaging technology, such as in-situ
and real-time observation, high sensitivity, and low damages to
biosamples [17–19]. Rhodamine dyes including tetramethylrho-
damine (TMRM) and rhodamine 123 have been used for visualizing
D
Wm and visualizing mitochondrial depolarization are of
Synthesis of 2,20-((1E,10E)-(piperazine-1,4-diylbis(4,1-pheny
lene))bis(ethene-2,1-diyl))bis(1-methylquinolin-1-ium)
iodide
D
(DPEI). The compound 2 was synthesized according to our previ-
ous report. Compound 1 (1 mmol) and 2 (3 mmol) was added into
8 mL ethanol in a round-bottom flask, which was stirred for more
than 5 min to mix evenly. 3 drops of piperidine was added into the
flask as catalyst, and the reactant was heated to reflux for 36 h. The
system was cooled down to room temperature and there were
solids precipitated. The solids were collected via filtration and
obtained by column chromatography. The final product presented
in brown powder (Yield, 41%). 1H NMR (400 MHz, DMSO d6) d 8.90
(d, J = 9.0 Hz, 2H), 8.56 (d, J = 9.1 Hz, 2H), 8.49 (d, J = 9.0 Hz, 2H),
8.34–8.20 (m, 4H), 8.13 (t, J = 8.0 Hz, 2H), 7.92 (dd, J = 12.6,
8.1 Hz, 6H), 7.69 (d, J = 15.5 Hz, 2H), 7.14 (d, J = 8.6 Hz, 4H), 4.50
(s, 6H), 3.65 (s, 8H). 13C NMR (101 MHz, DMSO) d 179.18, 164.64,
147.88, 144.19, 143.14, 141.13, 139.87, 136.69, 135.97, 129.44,
128.19, 127.89, 124.63, 123.24, 118.95, 116.96, 114.20, 114.17,
112.13, 106.39, 51.28, 45.67, 27.29, 13.82, 8.83. HRMS (m/z):
[M]2+ calcd. for C40H38N24+, 287.1543; found, 287.1543.
l
D
D
D
D
Wm under fluorescence microscopy [20,21]. The rhodamine dyes
can light up mitochondria with high Wm in live cells, and the flu-
D
orescence significantly decreased during mitochondrial depolar-
ization. Aggregation-induced emission (AIE) dyes were also
exploited for visualization of
co-workers have developed
emissive AIEgen for visualizing
DWm in off–on manner. Tang and
a
positively changed and red-
D
Wm, which displayed decreased
emission in mitochondria upon mitochondrial depolarization
[22]. Yoon’s group has attached triphenylphosphonium to
a
3. Results and discussion
green-emissive AIEgen, for detection of mitochondrial depolariza-
tion by reduced emission [23]. However, these probes might be
interfered by many factors, such as the inhomogeneous distribu-
tion of probe molecules and fluctuation of equipment status.
To avoid the interferences, fluorescent probes with subcellular
migration properties have been developed in recent years, for the
visualization of mitochondrial depolarization. Yu’s group has con-
structed a fluorescence DNA binder enabling migration from mito-
chondria to nucleus upon mitochondrial depolarization. Tian and
co-workers have developed fluorescent Zinc complex which could
migrate from mitochondria to nucleolus during the decrease of
3.1. Design and synthesis of the fluorescence probe
The immigration property was initially in consideration to
design the fluorescent probe. To achieve the
migration from mitochondria to nucleus/nucleolus, the probe
should meet two criteria. Firstly, the probe should possess cationic
DWm-dependent
groups to target mitochondria dependent on
DWm. Secondly, the
probe should display suitable affinity to nucleic acids (NA), which
ensures that the probe can move into nucleus/nucleolus after mito-
chondrial depolarization. Based on the two considerations, methyl
quinolinium group was selected as the targeting group. The posi-
tive charge can drive the probe into mitochondria in live cells.
The quinolinium group with short sidechain may afford high affin-
ity to NA by binding to the grooves.
Aniline group with strong electronic donating property was
selected to construct the other part of the probe. The strong elec-
tronic donor and acceptor endow probe DPEI a D-p-A electronic
D
Wm [24]. Our group has also developed fluorescent probes cap-
able of migration from mitochondria into nucleus/nucleolus with
the decrease of Wm levels [17,25]. Recently, our group has
D
reported a pH-sensitive RNA binder, which responsive to mito-
chondrial depolarization in ratiometric manner and via the subcel-
lular immigration from mitochondria to nucleolus [26]. Moreover,
we have also constructed a ratiometric probe visualizing mito-
chondrial depolarization based on intermolecular
p-stacking inter-
structure with long-wavelength emission. The probe was synthe-
sized based on the synthetic route shown in Scheme 1b, and the
synthesis procedures and characterizations were presented in the
Experimental Section and supporting information.
actions [27]. Yet, red-emissive fluorescent probes enabling
D
Wm-
dependent subcellular migration from mitochondria into nucleus/
nucleolus were still rarely reported. Since red-emissive probes
for bioimaging applications display unique advantages including
low interferences from endogenous fluorophores (typically
~450 nm from NADH in animal cells) [28], low photo-toxicity,
and high penetration depth. Therefore, it is an urgent task to
develop red-emissive fluorescent probes with subcellular migra-
tion properties for the visualization of mitochondrial
depolarization.
3.2. Optical properties of the probe
The optical spectra of the probe, including absorption and emis-
sion spectra in different solvents, were acquired initially as
depicted in Fig. 1. As demonstrated in Fig. 1a, the probe displayed
absorption in the range from 400 nm to 650 nm, with absorption
peaks around 500 nm in most solvents, and 550 nm in dichloro-
methane (DCM). The longer absorption in DCM should be resulted
from the halogen bond. Meanwhile, in Fig. 1b DPEI showed emis-
sion in the range of 550–750 nm with the fluorescence peak
around 630 nm, demonstrating the red-emissive property of the
probe in various solvents. It should be noted that the probe pre-
sented evidently enhanced emission in glycerol with high viscos-
ity, indicating that DPEI was sensitive to environmental viscosity.
To demonstrate the viscosity-sensitive fluorescence of the probe,
the emission spectra of DPEI in mixed solvent of glycerol and etha-
nol were obtained. As demonstrated in the Fig. S1, with the
increase of glycerol fraction and viscosity, the fluorescence of DPEI
steadily enhanced. Density functional theory (DFT) calculation was
thereafter performed, to understand its response to environmental
viscosity. As shown in Scheme 1a, the electron density in the ani-
2. Experimental section
Synthesis of 4,40-(piperazine-1,4-diyl)dibenzaldehyde (1).
Piperazine (10 mmol) was added into 40 mL methoxyethanol in
a flask, which was stirred for 10 min to mix evenly. Then p-
fluorobenzaldehyde (30 mmol) was dropped in, and the reactant
was refluxed for 24 h to finish the reaction. After cooled down to
room temperature, the mixture was mixed with 300 mL water
and extract with 300 mL dichloromethane twice. The dichloro-
methane layer was dried with anhydrous magnesium sulfate and
distilled in a rotary evaporator. The product was obtained by col-
umn chromatography and presented in light yellow powder (Yield,
36%). 1H NMR (400 MHz, DMSO d6) d 9.74 (s, 2H), 7.97–7.48 (m,
4H), 7.26–6.72 (m, 4H), 3.61 (s, 8H).
2

D. Guo, J. Sun, M. Tian et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119686
Scheme 1. (a) The chemical structure and frontier orbitals of probe DPEI; (b) The synthetic route of DPEI.
line moiety was higher than the quinolinium part in HOMO, while
the electron density in aniline moiety was lower in LUMO. These
results indicated the charge transfer from aniline moiety to quino-
linium group during excitation. The intramolecular charge transfer
(ICT) process in DPEI may cause the intramolecular motion, which
should be responsible for the weak emission in low-viscosity sol-
vents. In glycerol with high viscosity, the intramolecular motions
could be restricted, which can recover the fluorescence.
enhanced emission in high-viscose conditions, due to the inhibi-
tion of intramolecular motions. The bind of DPEI to DNA/RNA
might also inhibit the intramolecular motions and brought fluores-
cence enhancement.
Cytotoxicity of DPEI. To check the biocompatibility of DPEI, the
cytotoxicity of the probe was evaluated via (3-(4,5-dimethylthia
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As illus-
trated in Fig. S2, after incubation by 2 lM DPEI for 2 h, 24 h, and
Since the probe DPEI was expected to be a NA ligand that dis-
played high affinity to NA. The fluorescence of the probe in the
presence of DNA and RNA was then testified. As shown in Fig. 1a,
DPEI displayed decreased absorbance and slightly blue-shifted
absorption in aqueous conditions. Meanwhile, as shown in
Fig. 1b–d, the probe displayed very weak and red-shifted emission
peaked around 720 nm in buffer solutions. These optical characters
were in accordance with the H-aggregation, implying that DPEI
might form H-aggregates in aqueous solutions. After the introduc-
tion of DNA/RNA, the probe could bind to DNA/RNA and form
monomers, which may cause the fluorescence enhancement.
Moreover, as demonstrated above, DPEI exhibited evidently
36 h, the cell viability of live HeLa cells was 0.995, 0.981, and
0.952, respectively. These results demonstrated ignorable cytotox-
icity and high biocompatibility of probe DPEI, which was thus suit-
able for bioimaging experiments.
3.3. Different localizations of the probe in live and dead cells
Live HeLa cells were incubated with the probe, and cell images
were obtained by confocal microscope. As shown in Fig. 2, the flu-
orescent signals of DPEI were localized in extra-nuclear space of
live cells, and imaged as filament morphologies. These results
demonstrated that DPEI might stain mitochondria of live cells
3

D. Guo, J. Sun, M. Tian et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119686
Fig. 1. The absorption (a) and fluorescence (b) spectra of 5 lM probe DPEI in various solvents; the emission spectra of 5 lM probe DPEI in different concentration of DNA (c)
and RNA (d). Excitation wavelength: 500 nm; slit width: 5.0 for excitation, 5.0 for emission collection.
because of high
DWm. The DWm was completely depolarized in
fixed cells, and consequently HeLa cells were fixed by
paraformaldehyde and incubated by the probe. As shown in
Fig. 2, the probe targeted nucleolus and cytoplasm in fixed cells.
The obviously different localizations of DPEI in live and dead cells
could be used to monitor the mitochondrial depolarization. The
localization in nucleolus in fixed cells may be attributed to the
affinity of DPEI to NA, as illustrated in Fig. 1c and d. Since almost
all of the DNA distributed in the nucleus in eukaryotic cells, while
the RNA distributed in nucleolus and cytoplasm, DPEI may bind to
intracellular RNA in fixed cells. To clarify the targets of DPEI in
fixed cells, DNA and RNA digestion experiments have been carried
out as shown in Fig. S3. As shown in the figure, DPEI showed
intense emission in cytoplasm and nucleolus in fixed cells. After
the treatment of DNase and the digestion of DNA, the fluorescence
slightly decreased. By contrast, By contrast, after the treatment of
RNase and the digestion of RNA, the intracellular emission evi-
dently decreased. Thus, the probe should bind to RNA in fixed cells,
and the localization of the probe in nucleolus should be attributed
to its affinity to RNA.
Fig. 2. The DIC and fluorescence images of live and dead HeLa cells incubated by
M DPEI for 20 min. Excitation wavelength: 561 nm. Bar = 20 m.
2
l
l
Colocalization experiments was then used to confirm the loca-
tion of the probe in live cells. MitoTracker deep-red (MTDR), a
commercialized mitochondrial probe, was selected for co-
localization experiments. The fluorescence of MTDR could not
excited by 488 nm, while the probe DPEI showed no absorption
at 647 nm. Consequently, the fluorescence excited by 647 nm
was from MTDR, while that excited by 488 nm was from DPEI.
As illustrated in Fig. S4, the emission signal of DPEI overlapped
well with that of MTDR, and the Pearson’s co-localization coeffi-
cient was up to 0.92. The co-localization experiments further con-
firmed that DPEI targeted mitochondria in live cells.
3.4. Monitoring mitochondrial depolarization from subcellular
migration
As presented above, the probe DPEI was concentrated in mito-
chondria of live cells due to high
lus of dead cells with depolarized
potential to image the depolarization process of
D
D
Wm, and distributed in nucleo-
Wm. Consequently, DPEI was
Wm. Carbonyl
D
cyanide 3-chlorophenylhydrazone (CCCP) is a kind of protono-
phore that can rapidly and efficiently induce mitochondrial depo-
larization. The possible effect of CCCP on the probe DPEI was
4

D. Guo, J. Sun, M. Tian et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 255 (2021) 119686
in Fig. S5. The figure demonstrated that 10 mM H2O2 would not
bring significant emission change of DPEI, indicating that the inter-
action between H2O2 and DPEI could be ignored. Consequently,
live HeLa cells were pre-incubated by the probe DPEI, and then
treated with H2O2. As presented in Fig. 4, the probe DPEI stained
mitochondria of live cells. After the introduction of H2O2, the fluo-
rescence in mitochondria gradually faded, and the nucleolus was
steadily lighted up by intense emission. The change of probe local-
ization indicating the depolarization of
DWm induced by oxidative
damage. Therefore, the oxidative damage of HeLa cells induced by
H2O2 could monitored by the probe.
4. Conclusion
In summary, a red-emissive probe that can migrate from mito-
chondria to nucleolus during mitochondrial depolarization was
fabricated in this work. The probe exhibited a D-p-A electronic
structure, and displayed red emission peaked around 630 nm.
Meanwhile, the probe exhibited two positive charges and showed
high affinity to NA. As a result, the probe targeted mitochondria in
Fig. 3. The fluorescence images of HeLa cells pre-incubated by 2
20 min and then 10 lM CCCP was added to depolarized mitochondria. Bar = 20 lm.
l
M DPEI for
live cells with high
DWm, and immigrated into nucleolus upon
firstly assessed via fluorescence spectroscopy. As shown in Fig. S5,
Obviously, the addition of CCCP would not affect the fluorescence
of DPEI, indicating ignorable interactions between CCCP and DPEI.
Moreover, to prove that CCCP can actually depolarize the mito-
chondria, live HeLa cells were incubated by JC-1, a commercialized
mitochondrial depolarization. Consequently, mitochondrial depo-
larization could be efficiently monitored by the subcellular immi-
gration of the probe with high sensitivity. Particularly, the probe
displayed duple cationic groups and thus showed fast response
to mitochondrial depolarization with evidently elevated sensitiv-
ity. Oxidative damage by H2O2 to live cells was also successfully
monitored by the probe. Therefore, this red-emissive probe for
fluorescent probe to detect
and red fluorescence could be observed in live cells incubated by
JC-1. After the addition of 10 M CCCP for 5 min, the green emis-
sion increased while the red fluorescence decreased. These results
demonstrated that JC-1 can cause the rapid decrease of Wm in
DWm. As shown in Fig. S6, bright green
l
mitochondrial depolarization could be utilized to visualize
detect cell damage, and promote researches in relative areas.
DWm,
D
live cells. Live HeLa cells were pre-incubated by probe DPEI and
then treated by CCCP. As shown in Fig. 3, DPEI targeted mitochon-
CRediT authorship contribution statement
dria in cells with the high levels of
morphologies. After the addition of 10
gradually depolarized, and the DPEI molecules moved out of mito-
chondria and migrated into the nucleolus. Within 2.0–2.5 min, the
filament morphologies in cytoplasm disappears, and the emission
in nucleolus became intense. These results demonstrate that DPEI
D
Wm, and presented in filament
M CCCP the Wm was
Dingyi Guo: Methodology, Visualization, Investigation. Jie Sun:
Methodology, Visualization, Investigation. Minggang Tian: Con-
ceptualization, Validation, Writing - review & editing. Weiying
Lin: Conceptualization, Validation, Writing - review & editing.
l
D
Declaration of Competing Interest
could image the depolarization of
DWm with high sensitivity.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
3.5. Monitor oxidative damage by H2O2
Hydrogen peroxide (H2O2) is a kind of reactive oxygen species,
and also an important byproduct of respiration reactionꢂH2O2 can
bring severe oxidative damage to live cells and cause the mito-
chondrial depolarization. The possible interaction between H2O2
and DPEI was also testified using fluorescence spectra as shown
Acknowledgements
For financial support, we thank the National Natural Science
Foundation of China (NSFC) (21672083, 21877048, 21804052,
22077048), the Taishan Scholar Foundation (TS201511041), the
Natural Science Foundation of Shandong Province (ZR2018BB058),
and the startup funds of University of Jinan (309-10004,
160100331).
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2021.119686.
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