A bispyrene derivative as a selective fluorescent probe for RNA

Article abstract of DOI:10.1039/c3cc49430f

A novel bispyrene compound was synthesized to selectively detect RNA through excimer emission "turn-on" in aqueous solution at physiological pH (7.4). The compound was used to successfully image RNA in HeLa cells.

Full text of DOI:10.1039/c3cc49430f

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A bispyrene derivative as a selective fluorescent
probe for RNA†‡
Cite this: Chem. Commun., 2014,
0, 2505
5
a
a
a
b
b
Yifan Liu, Eun Jin Jun, Gyoungmi Kim, Ae-Ree Lee, Joon-Hwa Lee* and
a
Received 17th December 2013,
Accepted 13th January 2014
Juyoung Yoon*
DOI: 10.1039/c3cc49430f
www.rsc.org/chemcomm
A novel bispyrene compound was synthesized to selectively detect
RNA through excimer emission ‘‘turn-on’’ in aqueous solution at
physiological pH (7.4). The compound was used to successfully image
RNA in HeLa cells.
1
Since their discovery in 1868, ribonucleic acids (RNAs) have attracted
Scheme 1 Synthesis of fluorescent probe 1.
great interest due to their critical roles in biological processes.
RNA is known to perform active roles in the coding, regulation,
2
and expression of genes, among other functions. Consequently,
For the synthesis of probe 1, 1-hydroxypyrene-2-carbaldehyde
the development of RNA detection and recognition technology is (compound 2) was first prepared from 1-bromopyrene in 3 steps
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of great importance. Although there have been many reports on following a previously reported procedure. Compound 2 was then
3
4
fluorescent detection of DNA, G-quadruplex DNA, and nucleo- reacted with diethylenetriamine to give 1 as a red solid in 80%
5
,6
7–9
1
tides, relatively few examples of RNA detection can be found.
yield (Scheme 1). Probe 1 was fully characterized by H NMR,
C NMR and HRMS (FAB).
1
3
These probes for RNA detection often require complicated
synthetic steps to link the oligonucleotides to fluorophores. In
Fluorescence properties were examined in 99.8% Tris-HCl
addition, the selectivity for RNA over other nucleic acids is often buffer (and 0.2% DMSO) at pH 7.4. The emission spectra are
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insufficient. Recently, Chang et al. and Kim et al. reported the presented in Fig. 1. Upon the addition of 1.75 eq. of tRNA, ssRNA,
development of RNA-selective probes based on small positively ctDNA, UTP, TTP, poly (dA-dT) or poly (dG-dC) to probe 1, no
charged molecules. Chang and co-workers reported two small change, including any quenching effect, was observed in the
molecules for RNA detection; however, relatively small differences emission of probe 1. On the other hand, when 1.75 eq. of total
7
in affinity were observed between RNA and DNA. On the other RNA was added, a selective fluorescence enhancement was
hand, Kim et al. recently reported small cyclophane-containing observed at 470 nm, the excimer emission of pyrene fluoro-
1
0
imidazolium moieties which can selectively detect RNA over DNA phores. Fig. 1(b) presents the fluorescence titrations of probe
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in living cells.
1 with total RNA. As shown in Fig. 1(b), the peak at 470 nm
As delineated above, selective fluorescent detection of RNA
is still a challenging task. Here we report a neutral bispyrene
probe with high selectivity and sensitivity for RNA in aqueous
solution at physiological pH, 7.4, which can be used for fluorescence
imaging of RNA in live cells.
a
Department of Chemistry and Nano Science, Global Top 5 Research Program,
Ewha Womans University, Seoul 120-750, Korea. E-mail: jyoon@ewha.ac.kr;
Fax: +82-2-3277-3419; Tel: +82-2-3277-2400
b
Department of Chemistry and RINS, Gyeongsang National University, Jinju,
Gyeongnam 660-701, Korea. E-mail: joonhwa@gnu.ac.kr
†
‡
This paper is dedicated to Seiji Shinkai on the occasion of his 70th birthday.
Electronic supplementary information (ESI) available: Experimental details and
Fig. 1 (a) Fluorescence changes of 1 (20 mM) upon the addition of RNA,
tRNA, ssRNA, ctDNA, UTP, TTP, poly (dA-dT), poly (dG-dC) (1.75 equiv.
each) in 0.05 M Tris-HCl (pH 7.4). (b) Fluorescence titrations of 1 (20 mM)
with total RNA (0.0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75 equiv.) (lex = 348 nm).
supplementary figures and characterization of compounds. See DOI: 10.1039/
c3cc49430f
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1
Fig. 2 H NMR titration of 1 (1 mM) in DMSO-d
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with 0.0, 0.5, 1.0 equiv.
O.
Fig. 3 Fluorescence images of probe 1 in HeLa cells. Cells were fixed with 4%
paraformaldehyde and treated with RNase or DNase in the 40 mM Tris-HCl,
RNA (from bottom to up). RNA was dissolved in D
2
2
pH 7.5, 5 mM MgCl buffer for 30 min at 37 1C. And the cells were incubated in
the absence or presence of 20 mM JEJ-1 for 1 h at 37 1C. DRAQ5 was used as
arose from excimer emission of pyrene enhanced by increasing DNA stain. (a) 1 only, (b) DRAQ5, (c) merge, (d) DIC. Scale bars: 10 mm.
amounts of total RNA, which may indicate that the two pyrenes
in probe 1 are mostly p-stacked in total RNA.
Total cellular RNA consists of ssRNA, tRNA, rRNA and
microRNA. As shown in Fig. 1, excimer peak enhancement was
not observed for ssRNA or tRNA, unlike for total RNA. Thus, one
can propose that rRNA, accounting for B80% of total RNA,
induces the excimer peak enhancement. Fig. 2 presents the
1
H NMR spectra of probe 1 with total RNA. The aromatic protons
of the pyrene moieties were assigned based on 2D TOCSY and
ROESY experiments as shown in Fig. S4 (ESI‡). After adding
1
equiv. of total RNA, aliphatic protons at 3.79 and 3.04 showed
small downfield shifts to 3.83 and 3.10, respectively (Fig. 2).
In addition, the H10 resonances of the pyrene ring (doublet, 8.29
and 8.32) slightly shifted upfield to 8.29 and 8.26 ppm (Fig. 2).
However, the other aromatic protons showed no significant
chemical shift changes (Fig. 2). These results can be attributed
Fig. 4 Fluorescence intensities of probe 1 according to different serum
concentrations. The HeLa cells were cultured in 1% fetal bovine serum,
RPMI 1640 media for 24 h and then exchanged 10% fetal bovine serum,
RPMI 1640 media for 8 h. After 1 h incubation with 1 and 10 min incubation
with DRAQ5, the cell images were captured using a confocal microscope.
to the possible hydrogen bonding interaction between the OH (a) 1 only, (b) DRAQ5, (c) merge, (d) DIC. Scale bars: 10 mm.
group and the phosphate backbone of RNA. When the unique
conformation of rRNA induces p–p stacking interaction between
two pyrenes in probe 1 through intermolecular hydrogen bonding weak fluorescence, the cells cultured with 10% FBS displayed
interaction, the probe 1 exhibits fluorescence emission.
To test the RNA imaging properties of probe 1, HeLa cells that the live cell membrane is permeable to probe 1.
human epithelial adenocarcinoma) were incubated with probe Actinomycin D is an antineoplastic antibiotic that inhibits
(20 mM in 0.2% DMSO) in the culture media at 37 1C for 1 h. In cell proliferation. This compound inhibits the proliferation of
strong fluorescence (Fig. 4). In addition, these data confirm
(
1
the absence of probe 1, the cells exhibited no fluorescence, but cells nonspecifically by forming a stable complex with double-
the cells incubated with probe 1 exhibited strong fluorescence stranded DNA (via deoxyguanosine residues), thus inhibiting
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À1
(
Fig. 3). To determine whether probe 1 binds to DNA or RNA, the DNA-primed RNA synthesis. Actinomycin D (2 mg ml ) was
cells were fixed and treated with DNase or RNase. When the cells administered to cells for 4 h, and the fluorescence intensity of
were treated with RNase, no fluorescence was observed; however, probe 1 in these cells was measured. The fluorescence intensity was
cells treated with DNase exhibited fluorescence, as shown in reduced in cells receiving actinomycin D treatment, supporting the
Fig. 3. DRAQ5 stain was used as a DNA staining agent.
finding that probe 1 binds to RNA specifically (Fig. 5).
Serum (FBS, fetal bovine serum) in the culture media is
The potential cytotoxic effects of probe 1 were also examined
1
1
essential to cellular growth and RNA synthesis. Cells were in HeLa cells (Fig. S6, ESI‡). Cells were incubated with each
cultured with 1% FBS in RPMI-1640 media for 24 h, and then a concentration of probe 1 for 4 h and 24 h. Cell death was not
subset thereof was cultured with 10% FBS in RPMI-1640 media detected after 4 h of incubation, but 83% cell death occurred
for an additional 8 h. After probe 1 was administered to the live after 24 h incubation with 50 mM probe 1. This result can be
cells for 1 h, the cells were examined by confocal microscopy. attributed to the binding of RNA by probe 1 causing inhibition
While the cells cultured only with 1% FBS displayed relatively of RNA translation or degradation.
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