A novel aggregation-induced emission fluorescent probe for nucleic acid detection and its applications in cell imaging

Article abstract of DOI:10.1016/j.bmcl.2014.02.071

A new kind of aggregation-induced emission compound was synthesized and used as the probe of nucleic acid. The characterization of this compound was studied. Both the RNA and DNA were detected by using this probe. And the detection scope of DNA and RNA wa

Full text of DOI:10.1016/j.bmcl.2014.02.071

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1654–1656
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel aggregation-induced emission fluorescent probe for nucleic
acid detection and its applications in cell imaging
Xiaowei Xu , Shengyong Yan , Yimin Zhou, Rong Huang, Yuqi Chen, Jiaqi Wang, Xiaocheng Weng,
⇑
Xiang Zhou
College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, Wuhan University, Hubei, Wuhan 430072, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new kind of aggregation-induced emission compound was synthesized and used as the probe of nucleic
acid. The characterization of this compound was studied. Both the RNA and DNA were detected by using
this probe. And the detection scope of DNA and RNA was different. We researched the selectivity of our
probe in double and single strand DNA sequences. The visualization of gel electrophoresis and the cell
nucleus imaging were researched as well. Compared with the traditional nucleus dye Hoechst 33258,
our probe also has the potential to be nucleus dye. And the cell toxicity was well performed by MTT
assays.
Received 4 February 2014
Revised 19 February 2014
Accepted 25 February 2014
Available online 5 March 2014
Keywords:
Fluorescent
Nucleic acid
Ó 2014 Elsevier Ltd. All rights reserved.
Aggregation-induced emission
Gel electrophoresis
Cell imaging
Dye
It is well known that the detection of nucleic acid is very impor-
tant in genetic engineering, forensics, and bioinformatics.1–3 There-
fore, many nucleic acid probes have been designed and synthesized
based on the structures of nucleic acids. It has been reported that
ethidium bromide (EB), Hoechst dyes, acridizinium salts, cyanine
derivatives, and ruthenium complexes which based on fluorescent
enhancement have been developed as nucleic acid probes.4–15
There into, EB is cheap and widely used in molecular biology labo-
ratories as a nucleic acid stain. However, EB is thought to be a
strong mutagen or carcinogen. Currently, some alternatives to EB
such as SYBR-based dyes are found to be less carcinogen.14 So it
is desirable to develop more fluorescent probes which could be
safer for detection of nucleic acid in aqueous solution.
of aggregation-induced emission (AIE) that the fluorescence
enhancement attributed not only to the spatial confinement effect
but also to the formation of specific supramolecular stacking archi-
tecture is the opposite of ACQ effect and has been widely studied re-
cently.1–3,22 We studied the AIE characteristic of FcPy by using the
traditional methods (Fig. S8).
nucleic acid
FcPy
Some fluorescent dyes can aggregate in aqueous buffer or be
bound to biomacromolecules.16–18 The fluorescent signals fade
when the dyes aggregate. This aggregation-caused quenching
(ACQ) effect is a major problem in the development of bioprobes
and biosensors.19–21
strong fluorescent
weak fluorescent
Herein we designed and synthesized
a
derivative of
p-phenylenediacetonitrile for the detection of nucleic acid. And we
named the compound as FcPy. FcPy has strong fluorescence emis-
sion under UV light (kex = 365 nm) (Scheme 1). This phenomenon
N
Br
NC
UV
O
O
CN
365 nm
N
Br
FcPy
⇑
Corresponding author.
E-mail address: xzhou@whu.edu.cn (X. Zhou).
These authors contribute equally to the works.
Scheme 1. Schematic illustration of the fluorescence ‘Turn-On’ system for detec-
tion of nucleic acid with FcPy.
http://dx.doi.org/10.1016/j.bmcl.2014.02.071
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

X. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1654–1656
1655
1000
900
800
700
600
500
400
300
200
(a)
800
600
400
200
0
425
450
475
500
525
550
575
0
200 400 600 800 1000 1200 1400 1600
Time / s
wavelength (nm)
Figure 1. Real-time emission intensity changes of FcPy (100 lM) at 505 nm upon
single-strand DNA (1 lM, 75 nt). kex = 396 nm.
900
800
700
600
500
400
300
200
100
0
(b)
700
(a)
600
500
400
300
200
100
0
=
−
+
+
0
500
1000
1500
g/ml)
2000
2500
RNA (
µ
Figure 3. (a) Changes in the emission spectrum of FcPy (5 lM) upon the different
450
500
550
600
650
concentrations (0–2.5 mg) of RNA in PBS. kex = 396 nm, kem = 420–575 nm. (b) Plot
of I/I0 À 1 at 470 nm versus the RNA concentration. I0 = emission intensity in the
absence of RNA.
wavelength (nm)
(b)
6
The results indicated that FcPy had almost no selectivity between
single and double strand DNA as the intensity of double strand
DNA was almost double of single strand DNA when interacted with
FcPy. Next, we used S1 nuclease to digest the DNA sequence and
recorded the fluorescence emission. From Figure S4 we can see that
the fluorescence intensity was nearly the same as control after
digesting with S1 nuclease. This is because that when the DNA
sequence is digested into nucleotides, the aggregated fluorescent
probes adhered to DNA sequence is also dispersed into the aqueous
solution. This also proved that FcPy has the property of AIE effect.
In order to study the kinetics of FcPy and nucleic acid interac-
tion, we recorded the real-time emission intensity of FcPy
5
4
3
2
1
=
−
+
+
0
0
5
10
15
20
25
30
ctDNA (µg/mL)
(100 lM) at 505 nm upon single-strand DNA (1 lM, 75 nt) with
excitation at 396 nm. The fluorescence intensity went to plateau
Figure 2. (a) Changes in the emission spectrum of FcPy (5
lM) upon the different
concentrations (0–30 g) of ctDNA in PBS. kex = 396 nm, kem = 420–600 nm. (b) Plot
l
at about 20 min (Fig. 1).
of I/I0 À 1 at 475 nm versus the ctDNA concentration. I0 = emission intensity in the
According to the results mentioned above, we tried to detect
natural DNA and RNA with FcPy. We chose ctDNA and RNA from
torula yeast as the target nucleic acid since they are cheap and easy
to obtain. FcPy was dissolved in phosphate-buffered saline (PBS)
solution (10 mM, pH 7.4), and the concentration of FcPy was
10 mM. The fluorescence intensity recorded at 475 nm is enhanced
quickly when the concentration of ctDNA is low, but it is gradually
saturated when the concentration of ctDNA becomes higher
(Fig. 2a). There is a gradual blueshift of the emission maximum
from 505 to 475 nm, this was elucidated by Park and coworkers.22
absence of ctDNA.
Firstly, we investigated the interaction of FcPy with nucleic acid
and single strand DNA sequences were chosen as the model. We
found that the fluorescence emission was brighter with increasing
length of the DNA sequences (Fig. S1). This was consistent with AIE
effect. As FcPy can be cations in aqueous media and DNA se-
quences have negative charges, the longer sequences have more
negative charges to be adhered with more FcPy.
Then we attempted to explore whether FcPy has an ability of
selectivity in the detection of double strand DNA and single strand
DNA. We tested the fluorescence emission of a single strand DNA
and its double strand DNA (Fig. S2) after interaction with FcPy.
This is because that there two forms of
in different situations. The change of (I/I0 À 1) versus ctDNA con-
centration (0–30 g/mL) was fitted well to the Boltzmann function
with an R2 value of 0.9738 as shown in Figure 2b. As the same as
p–p overlap of the molecule
l

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X. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1654–1656
calculated out the IC50 of FcPy as 26.0 lM (Fig. S5). From Figure 5,
the fluorescence image of HeLa cells stained with FcPy was signif-
icantly bright. Then we stained the cells with Hoechst 33258 which
is commercial available, the merged image (Fig. 5c) shows that
FcPy could successfully stained the nucleus of cell as well as Hoe-
chst 33258.
In summary, we have successfully developed a new kind of
probe for the detection of nucleic acid. It could detect double
strand, single strand DNA and RNA. And it also can quantitatively
analyze the nucleic acids in solution and visualize the DNA bind
in gels. With the results of the cell imaging, FcPy might be used
as the potential nucleus dye.
1
2
3
4
5
6
7
8
Figure 4. Staining of single-strand DNA in PAGE by FcPy. Concentrations of DNA
(75 nt) in lanes 1–8: 0, 0.25, 0.5, 1.0, 10.0, 25.0 and 50.0 g (from left to right).
Concentration of dyes: 100 M. Staining time: 20 min.
l
l
Acknowledgments
This work was supported by the National Basic Research Pro-
gram of China (973 Program) (2012CB720600, 2012CB720603),
the National Science Foundation of China (Nos. 91213302,
21072115, 21272181), National Grand Program on Key Infectious
Disease (2012ZX10003002-014), Supported by Program for Chang-
jiang Scholars and Innovative Research Team in University
(IRT1030).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
02.071.
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as low as 1.0 lg (lane 4, Fig. 4).
At last, we employed FcPy in cell imaging. Before we performed
FcPy in cells, we tested the cytotoxicity of FcPy. Living HeLa cells
were incubated with various concentrations of FcPy for 48 h, after
which the percentages of the viable cells were quantified. We