Fluorescence behavior of a unique two-photon fluorescent probe in aggregate and solution states and highly sensitive detection of RNA in water solution and living systems

Article abstract of DOI:10.1039/c6cc03746a

It is found that 2,7-substituted carbazole derivative HVC-6 possesses distinct luminescence features in both aggregate and solution states. In view of this, probe HVC-6 realizes highly sensitive detection of RNA in pure water systems by an aggregation-disaggregation method for the first time.

Full text of DOI:10.1039/c6cc03746a

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DOI: 10.1039/C6CC03746A
In this work, we developed a unique fluorescent probe HVC-6 for high sensitive detecting RNA in pure water
system and living systems along with aggregation-disaggregation of probe for the first time.

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Fluorescence behavior of a unique two-photon fluorescent probe
in aggregate and solution states and high sensitive detection of
RNA in water solution and living systems†
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
b
a
Yong Liu, Fangfang Meng, Longwei He, Xiaoqiang Yu * and Weiying Lin *
DOI: 10.1039/x0xx00000x
www.rsc.org/
It is found that 2,7-substituted carbazole derivative HVC-6 for detecting RNA in water with aggregation-disaggregation of
possesses distinct luminescence feature in both aggregate and probe. We hope this probe possesses higher sensitive to RNA in
solution states. In view of this, the probe HVC-6 realizes high pure water system and living system than conventional RNA probe
sensitive detecting RNA in pure water system by aggregation- (Scheme 1).
disaggregation method for the first time.
In the past few years, the investigations of conventional small-
molecular dyes have made great contributions to the fundamental
1
understanding of luminescence processes at molecular level. The
conclusions drawn from the dilute solution data, however, cannot
2
commonly be extended to the concentrated solutions. Thus,
aggregation-induced emission (AIE) dyes were discovered
3
by Tang in 2001. Lipophilic AIE molecules form nanoaggregates in
aqueous solution spontaneously because of their hydrophobic
3a
nature. These nanoaggregates have been successfully applied for
detecting various biological analytes and show more excellent
properties than conventional biomaterials. Herein, we describe the
4
Scheme. 1 Strategies for the sensing of RNA
development of a unique fluorescent probe for high sensitively
monitoring RNA by aggregation-disaggregation method.
Toward this end, we herein introduce HVC-6 (Scheme. 2a) as the
Ribonucleic acid (RNA) was made from monomers known first fluorescent dye for high sensitive detecting RNA in pure water
5
as nucleotides. It played central roles in converting genetic by aggregation-disaggregation method. The key to the design is that
6
information from genes into the amino acid sequences of proteins.
this novel fluorescent dye should possess distinct luminescence
7
RNA was discovered by Friedrich Miescher in 1869, however, the feature in aggregate and solution states. As two-photon (TP)
RNA-specific dyes were first used to detect RNA until 1984. More fluorescence is more favorable than one-photon (OP) for imaging
recently, several classes of molecular probes have been developed applications in living systems, we selected the 2,7-position
for RNA detection in living cells. Molecular Probes Co. offers a substituted carbazole derivative as the TP platform. In general, the
8
11
9
commercially available RNA probe “SYTO RNA-Select” for RNA probe HVC-6 possessing A-π-D-π-A “Helical Structures’’ display
10
imaging in living cells. However, the investigations of RNA probes aggregate states and weak red fluorescence in aqueous due to twist
12
mainly focus on solution states and lacking investigation of probes. intramolecular charge transfer (TICT) (Fig. S1†). And similarly
Thus, the goal of our work is to design a unique fluorescent probe structure was capable of detecting nucleic acid based on
13
intercalative binding mechanism. Thus, we envision that the free
probe HVC-6 may exhibit red-shifted emission owing to aggregate
of probe. However, in the presence of RNA, the intercalative
binding of RNA to HVC-6 will decrease aggregation of probe and
induce a blue-shift in the emission profiles (Scheme 2b).
Chemical synthesis of HVC-6 is accomplished in a total of five
steps (Scheme 2a). The optimization of 4,4’-dibromo-2-
nitrobiphenyl (1) started from readily available 4,4’-
dibromobiphenyl and nitryl. 2,7-Dibromocarbazole (2) is steadily
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prepared as isolable intermediates for synthesize 2,7-dibromo-9-(2- emission color underwent further hypsochromically VshieiwftAr(tFicilge.O2nlbin)e,
ethoxyethyl)-9H-carbazole (3). HVC is obtained by Heck reaction however, fluorescent intensity is graduallyDbOeIc:o10m.1e03w9e/Ca6kC(CF0ig3.7246c)A.
between 3 and 4-vinylpyridine. Treatment of Iodine hexane with Moreover, solid fluorescence spectrum of HVC-6 further prove this
HVC in acetone results in the formation of probe HVC-6. The probe AIE features (Fig. 2d). The above results show that HVC-6
14
synthetic details of these compounds are shown in the Supporting should be a TICT luminogen with AIE property. Thus, we envision
Information.
the dye HVC-6 may form spherical nanoparticles in definite fw
values.
The aggregate characteristics of HVC-6 are studied by dynamic
light scattering (DLS) and transmission electron microscopy (TEM)
techniques (Fig. 2). In pure DMF solution, HVC-6 cannot form
aggregate states (Fig. 2a); In water/DMF (fw = 10%) mixtures
solution, the compound HVC-6 form abundant irregular aggregation
(
Fig. 2b). Moreover, in water/DMF (f = 90%) mixtures solution, the
w
probe HVC-6 forms spherical nanoparticles with radius 70-100 nm
Fig. 2c). Similar to results of TEM, DLS on 90/10 (fw = 90%)
water/DMF highlight spherical materials with an average size of 70-
00 nm (Fig. 2d). The above results further prove that HVC-6 is
(
1
luminogen material with AIE property. The results of TEM and DLS
techniques are consistent with optical properties of probe in
water/DMF mixtures (Fig. 1). We predict this material is capable of
detecting intracellular analytes by aggregation-disaggregation
method.
Scheme. 2 (a) Synthesis of probe HVC-6; (I) Fuming HNO , acetic
3
acid; (II) P(o-Et)3, Ar; (III) NaOH, DMF, after added 1-bromo-2-
ethoxyethane; (IV) Palladium(II) acetate, tri-o-tolylphosphine and
K CO , N-methyl-2-pyrrolidone and 4-vinylpyridine; (V) Acetone,
2
3
Iodine hexane, reflux; (b) Rational design of the novel TP probe
HVC-6 with distinct fluorescence signals in aggregate and solution
states.
Fig. 2 (a) TEM pictures of water/DMF (f = 0%), (b) water/DMF (f =
w
w
-5
1
0%) and (c) water/DMF (f = 90%) dispersions of HVC-6 at 2X10
w
M; (d) DLS measurements of HVC-6 dispersions in water/DMF (f =
w
-5
90%) mixtures at 2X10 M.
To check the probe HVC-6 can detect RNA in water solution by
aggregation-disaggregation
method,
fluorescence
titration
Fig. 1 (a) Photographs of HVC-6 in water/DMF mixtures were taken
under UV illumination; (b) Fluorescence spectra of HVC-6 in
water/DMF mixture; (c) Plots of maximum emission intensity (I) and
experiments are carried out. First, we set out to investigate the
spectral changes of HVC-6 with increasing RNA. As shown in Fig. 3,
when RNA increased to buffer solution of probe, the emission color
underwent further hypsochromically shift at 488 nm excitation (Fig.
wavelength (λem) of HVC-6 versus water/DMF (f , vol %) in
w
water/DMF mixtures; (d) Solid fluorescence spectra of HVC-6.
Concentration of HVC-6: 10 μM. λex = 488 nm.
3a and inset). This phenomenon is similar with photophysical
properties of probe in water/DMF mixtures. The fluorescence
titration experiments demonstrate that the fluorescence intensity
evidently increased at 550 nm with the addition of RNA and show
Solid HVC-6 shows strong red fluorescence at wide-field
excitation (Fig. S2†). We envision that the dye HVC-6 may possess
aggregate states in water/DMF mixtures with a high water fraction
25-fold enhancement (Fig. 3b). In addition, the probe HVC-6
present similar results at 800 nm excitation (Figs. 3c and d).
To prove the probe can disaggregation in the presence of RNA in
water, DLS experiment is carried out. In water solution
(
fw). To prove the above assumption, we examine fluorescence
behavior of HVC-6 in water/DMF mixtures . Photographs of HVC-6
in water/DMF mixtures are taken under UV illumination (Fig. 1a).
We systematically change the polarity by admixing polar DMF and
water, then we measure the emission spectra of HVC-6 in solvent
(
Concentration of HVC-6 was 10 μM), spherical nanoparticles with
the highest value of radius was 250 nm (Fig. S3†); However, in the
presence of RNA, radius of spherical nanoparticles decreased to 60
mixtures. When f increased from 0 (Φ=0.12) to 100% (Φ=0.01), the
w
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nm. The above results suggest that the probe HVC-6 can detect RNA toxicities of HVC-6 against HeLa cells. The cytotoxicity of the HVC-6
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DOI: 10.1039/C6CC03746A
probe is evaluated using the standard MTT assays, and the data
in water solution by aggregation-disaggregation method.
To prove sensitivity of probe, we will investigate detection limit indicate that the probe has low toxicity for the living cells (Table.
of the probe HVC-6. Under OP and TP excitation condition, the S1†). In fluorescent imaging, the red fluorescence of HVC-6 mainly
detection limit for HVC-6 is calculated to be 0.2 μM (Figs. 4a and b), localizes at the cytoplasm and nucleoli accompanied with faint
however, the detection limit of commercially available probe SYTO nuclei distribution rather than cell nucleus. As we all know, the
RNA-Select is 1.8 μM (Fig. S4†), indicating that the probe possesses nucleolus contains abundant proteins and RNAs, especially
9
9b
higher sensitivity than the existing RNA probes.
ribosomal proteins and rRNA. Red fluorescence signals of cells
loaded with probe may be caused by endogenous RNA cytoplasm
and nucleoli. To verify our assumption, the digest test of
ribonuclease (RNase) is carried out. As shown in Fig. S6†, RNase
only hydrolyzes the RNA in the cells and did not influence the DNA,
is performed. Moreover, the probe HVC-6 may exist in cells in the
form of aggregation after the digest test.
For RNA probes, generally, the interference of DNA in vitro is not
9e
ignored. Thus, fluorescence features of the probe should be
similar with commercially available probe SYTO RNA-Select in vitro
and in cells (Fig. 5a). For HVC-6 and SYTO RNA-Select, in vitro,
fluorescence response of probe to DNA and RNA in buffer solutions
has been investigated (Figs. 5b and c). In living cells, fluorescence of
RNA mainly from cytoplasm and nucleoli (Figs. 5d and e).
Fluorescence of two probes are stronger than that of DNA.
However, in situ imaging, the probe HVC-6 emits stronger
fluorescent emission than commercially probe SYTO RNA-Select at
the same conditions (Fig. S7†). The above results prove that HVC-6
can selective imaging RNA in living cells. But, different from SYTO
Fig. 3 (a) OP fluorescence spectra of HVC-6 (10 μM) in tris buffer
solution with the addition of RNA (0-550 equiv). Inset: Relative OP
fluorescence spectra of HVC-6 to buffer water solution in the
absence and presence of RNA. λex = 488 nm; (b) OP fluorescence
intensity changes at 570 nm of HVC-6 (10 μM) with the amount of
RNA. λ = 488 nm ; (c) TP fluorescence spectra of HVC-6 (20 μM) in RNA-Select,
e HVC-6 can high sensitively detect RNA by
ex
tris buffer solution with the addition of RNA (0-800 equiv). Inset: aggregation-disaggregation method.
Relative TP fluorescence spectra of HVC-6 to buffer water solution
in the absence and presence of RNA. λex = 800 nm; (d) TP
fluorescence intensity changes at 570 nm of HVC-6 with the amount
of RNA. λ = 800 nm.
ex
Fig. 4 Normalized response of the (a) OP and (b) TP fluorescence
signal by changing the concentration of RNA.
Furthermore, the probe HVC-6 is treated with various relevant
analytes including ions, reactive oxygen species, reducing agents,
small molecule thiols, proteins, polysaccharides and nucleic acid in
Fig. 5 (a) Fluorescence features of the probe HVC-6 and
tris buffer solution to investigate the selectivity (Fig. S5†). The
−
−
-
−
2−
commercially available probe SYTO RNA-Select in vitro and in living
cells; (b) Fluorescence imaging of living cells with HVC-6 (2 μM); (c)
Fluorescence responses (at 550 nm) of HVC-6 (2 μM) in the
presence of RNA and DNA. RNA and DNA concentration: 2.90 mM;
addition of the representative anions (Cl , HSO , I , NO and SO4 ),
3
2
+
2+
2+
2+
metal ions (K , Mg , Ca and Zn ), reactive oxygen and nitrogen
species (H O and NO ), and reducing agents (SO ) at relevant
−
2
2−
3
2
2
concentrations induced no marked fluorescence enhancement.
Furthermore, the small-molecule thiols such as glutathione (GSH),
homocysteine (Hcy), proteins and polysaccharides also elicited no
marked response. However, similar with conventional RNA small-
(
(
d) Fluorescence imaging of living cells with SYTO RNA-Select (5 μM);
e) Fluorescence responses (at 525 nm) of SYTO RNA-Select (2 μM)
in the presence of RNA and DNA, RNA and DNA concentration: 150
mg/mL. λex = 488 nm; Scale bar = 20 μm. Statistical analyses were
performed with Student’s t-test (n = 4). * P < 0.05.
9
molecular probes, the interference of DNA in vitro is not ignored.
In order to be useful as imaging agents, fluorescent probes
should have low cytotoxicity. Thus, we investigate the potential
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We further investigate interactions between HVC-6 and RNA by and photostability. We expect that the design stratVeiegwyAmrticilgehOtnlbinee
induced circular dichroism (CD) spectra. As shown in Fig. S8†, CD extended for development of a wide varietyDoOfI:T1P0.f1u0n3c9t/iCo6nCaCl p03ro74b6eAs
signal of RNA appear in range of 200-300 nm, and plus and minus for detecting different RNAs or specific RNA sequence by
two signals confirm spiral chain characteristics of RNA. Due to aggregation-disaggregation method.
induction of RNA, cotton peaks of the probe HVC-6 at 400 (-) and
70 (+) nm corresponded to its absorption peaks bound with RNA. 21172063), Taishan Scholar Foundation (TS 201511041), doctor
The results demonstrate that HVC-6 is capable of detecting nucleic start up fund of University of Jinan (160082102). NSFSP
Funding was partially provided by NSFC (51503077, 21472067,
4
acid based on intercalative binding.
(ZR2015PE001), and general start up fund of the University of Jinan.
Photostability is an important criterion measuring an excellent
probe. In vitro, the probe exhibits good one- and two-photon
photostability (Fig.S9†). Next, we investigate photostability in living
cells, commercially available RNA probe SYTO RNA-Select as control.
As shown in Fig. 6, SYTO RNA-Select exhibits significant
photobleaching with only 40% signal intensity remaining.
Fluorescence signals of HVC-6 only decreased 8%. The results prove
that the probe HVC-6 shows higher photostability than
commercially available RNA probe.
Notes and references
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Fig. 6 Comparison of photobleaching of HVC-6 and SYTO RNA-Select
in confocal fluorescence microscopy imaging. Cells stained with 5
μM probe for 30 min; λex = 488 nm; λem = 490-600 nm; Scale bar =
2
0 μm.
Chem. B, 2014,
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2, 2688; (e) G. Song, Y. Sun, Y. Liu, X. Wang,
In TP imaging, we have demonstrated that the probe can image
2
RNA in living cells at 800 nm excitation (Fig. S10†). To demonstrate
the utility of this probe tissues imaging, the mouse liver tissue slices
are treated with the probe HVC-6. All of these experiments are
performed in compliance with the relevant laws and institutional
guidelines, and are approved by the Animal Ethical Experimentation
Committee of Shandong University. And fluorescence images of the
tissue slices are acquired at 800 nm excitation. Significant
fluorescence is observed up to 100 μm in green channel (Fig. S11†).
Thus, taken together, the results demonstrate that the probe can
image RNA in living tissues.
7
1
1
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In summary, we have rationally engineered a novel TP
fluorescent probe HVC-6, which remarkably display two different
emission peaks to aggregate and solution states in the presence and
absence of RNA for the first time. And the probe shows sensitivity
1
1
3 T. Y Ohulchanskyy, H. E. Pudavar, S. M. Yarmoluk, V. M.
Yashchuk, E. J. Bergey, P. N. Prasad, Photochem. Photobiol.,
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excitation. These unique attributes enable the probe to be
employed to one- and two-photon image endogenous RNA in living
systems. In addition, compare with commercially available RNA
probe SYTO RNA-select, the probe HVC-6 exhibits higher sensitivity
1
4
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