A 2,7-carbazole-based dicationic salt for fluorescence detection of nucleic acids and two-photon fluorescence imaging of RNA in nucleoli and cytoplasm

Article abstract of DOI:10.1039/c1ob05123g

A new carbazole-derived dicationic compound, namely 2,7-bis(1-hydroxyethyl- 4-vinylpyridinium iodine)-N-ethylcarbazole (2,7-9E-BHVC), with a large two-photon action absorption cross section in nucleic acids has been obtained. Moreover, it possesses the potential of imaging RNA in nucleoli and cytoplasm in two-photon fluorescence microscopy and exhibits good counterstain compatibility with the commercial fluorescent nucleic dye DAPI.

Full text of DOI:10.1039/c1ob05123g

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A 2,7-carbazole-based dicationic salt for fluorescence detection of nucleic
acids and two-photon fluorescence imaging of RNA in nucleoli and cytoplasm†
Xin Liu,a Yuming Sun,b Yuanhong Zhang,a Fang Miao,a Guancong Wang,a Hongshi Zhao,a Xiaoqiang Yu,*a
Hong Liu*a and Wai-Yeung Wong*c
Received 22nd January 2011, Accepted 21st February 2011
DOI: 10.1039/c1ob05123g
A new carbazole-derived dicationic compound, namely
2,7-bis(1-hydroxyethyl-4-vinylpyridinium iodine)-N-ethyl-
carbazole (2,7-9E-BHVC), with a large two-photon action
absorption cross section in nucleic acids has been obtained.
Moreover, it possesses the potential of imaging RNA
in nucleoli and cytoplasm in two-photon fluorescence
microscopy and exhibits good counterstain compatibility
with the commercial fluorescent nucleic dye DAPI.
hydrophobic biosensors including nucleic acid (NA) probes may
also non-specifically bind with proteins and membranes in cells;
third, knowledge of the interaction mechanism between RNA and
fluorescent probes is still not enough compared with the wealth
of deeper understanding of DNA biosensors, including outside,
groove and intercalative binding.13–15 Thus it is necessary to find
new RNA fluorescent probes.
Recently, two-photon fluorescence microscopy (TPM) has
become an indispensable tool in many biological laboratories
due to its unique advantages in bioimaging16–19 and various
two-photon fluorescent probes have been investigated intensively
and extensively.20–24 To our knowledge, only one two-photon
RNA probe has been reported: Ohulchanskyy et al. found
that conventional NA dye cyan 40 can be used to image
RNA in nucleoli by TPM.25 However, its two-photon action
absorption cross section (U¥d), a crucial parameter for two-
photon fluorescent probes, is not reported. Recently, we have
reported a series of two-photon DNA and mitochondria probes
from carbazole-containing cationic compounds, especially 3,6-
carbazole-dicationic salts, they have been successfully used to
image nuclei in plant cells and tissues and mitochondria in
living cells by TPM.26,27 The works of Chang and ourselves
both demonstrated that structural changes in carbazole cationic
compounds can practically influence their cellular responses and
become different fluorescent probes.26–28 According to the idea
mentioned above, an isomer of 3,6-carbazole dicationic salt, 2,7-
carbazole dicationic salt (2,7-9E-BHVC), was synthesized (Chart
1), and our experimental results have shown that 2,7-9E-BHVC
is a two-photon turn-on fluorescent probe for NA, and its U¥d
are 1.67 GM and 30 GM in the absence and presence of NA,
respectively. Moreover, the staining results from both wide-field
fluorescence microscopy and TPM have shown that 2,7-9E-BHVC
is able to image the RNA in nucleio and cytoplasm.
Introduction
Both RNA and DNA are important biomacromolecules.1,2 How-
ever, compared to the many fluorescent probes imaging DNA,3–7
RNA probes for cell imaging are rarely reported. Molecular
Probes Co., which sells various DNA probes such as DAPI,
series of Hoechsts, and propidium iodide, only offered a classical
commercial RNA probe “SYTO RNA-Select” for imaging RNA
in living cells, but its chemical structure has not been described.8
Li and coworkers screened a large combinatorial library of 1336
fluorescent styryl molecules and only ultimately obtained three
RNA probes.9 Yu et al. reported a europium complex for nucleoli
imaging.10 At the same time, Turro’s team also constructed two
RNA probes by covalently linking ruthenium(II)11 or fluorescein12
to phenanthridine derivatives, which are the oldest and most
often used fluorophores intercalating with DNA or RNA. The
possible reasons why RNA probes are rare could be at least three-
fold: first, small nucleic-acid binding molecules generally have
better affinity for double-stranded DNA than for RNA; second,
aState Key Laboratory of Crystal Materials, Shandong University, Jinan,
250100, P. R. China. E-mail: yuxq@sdu.edu.cn, hongliu@sdu.edu.cn;
Fax: 0531-88366418; Tel: 0531-88366418
bSchool of Information Science and Engineering, Shandong University, Jinan,
250100, P. R. China
cAreas of Excellence Scheme (University Grants Committee, Hong Kong)
and Department of Chemistry, Hong Kong Baptist University, Waterloo
Road, Kowloon Tong, Hong Kong, P. R. China. E-mail: rwywong@
hkbu.edu.hk; Fax: (+ 852)3411-7348
† Electronic supplementary information (ESI) available: Experimental
details and characterization of 2,7-9E-BHVC, measurement equipment
and materials, measurement of two-photon absorption cross section,
cell culture and staining, wide-field fluorescence microscopy imaging
and two-photon fluorescence microscopy imaging experiment, UV–vis
absorption and fluorescence spectra of 2,7-9E-BHVC in various solvents,
photophysical properties of 2,7-9E-BHVC. See DOI: 10.1039/c1ob05123g
Chart 1 Chemical structure of 2,7-9E-BHVC.
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Results and discussion
The synthesis (Scheme S1†) and characterization of 2,7-9E-
BHVC are shown in the ESI.† Fig. S1† shows the solvatochromic
studies of 2,7-9E-BHVC. Its one-photon and two-photon fluo-
rescence intensities are high in organic solvents and very low
in aqueous solution. The appreciable solvent effect implies that
the intramolecular charge transfer state is predominant in the
photophysical behavior of 2,7-9E-BHVC, which is similar to our
other carbazole derivatives.26,27 Accordingly, the weak emission of
2,7-9E-BHVC in water can be attributed to the strong interaction
of the molecules at excited state with the water environment, which
induces the formation of twisting intramolecular charge transfer
state, resulting in the increase of nonradiative decay.26,27,29 On the
other hand, the self-aggregation of 2,7-9E-BHVC with low solubil-
ity in water could also result in self-quenching. It is well known that
the concentration-quenching effect is common for many aromatic
compounds due to the formation of sandwich-shaped excimers
and exciplexes aided by the collisional interactions between the
aromatic molecules in the excited and ground states.30,31
Subsequently, the interaction of 2,7-9E-BHVC with NA was
carefully studied (Fig. 1a). When the compound was mixed with
NA in a 100 : 1 [NA]/[dye] ratio, both DNA and RNA induced
marked hypochromism of the absorbance (ca. 18%) and red shift
(ca. 18 nm) of the absorption maximum, which suggests that the
dye interacts apparently with NA.13,14 Moreover, 2,7-9E-BHVC is
only weakly fluorescent in tris-HCl buffer, whereas its fluorescence
intensity shows an approximately 25 fold enhancement in NA. The
mechanism of the light-switch effect of the dyes binding to NA
could be explained by two factors: firstly, the restricted action
on the torsional motion of vinyl groups that is derived from
the interactions between the cationic N-pyridinium unit and the
anionic phosphate of NA; secondly, the dye is protected from
water in the hydrophobic environment offered by the higher-order
structure of NA. In addition, the gradual change of fluorescence
with addition of NA is shown in Fig. 1b, in which the fluorescence
of 2,7-9E-BHVC enhances sharply at the low ratio (<40 : 1
[NA]/[dye] ratio) and then tends to mildly increase until saturation
point is reached (from 60 : 1 to 180 : 1 [NA]/[dye] ratio). The
whole process of fluorescence enhancement with the addition of
RNA is identical to that of DNA. Furthermore, the two-photon
fluorescence spectra of 2,7-9E-BHVC in tris-HCl buffer, DNA
and RNA solutions were examined at 800 nm which is an optimal
excitation wavelength provided by a commercial mode locked
Ti:sapphire laser source (Fig. 2a). 2,7-9E-BHVC exhibits bright
two-photon induced fluorescence both in DNA and RNA and the
Fig. 2 a: Two-photon excited fluorescence spectra of 2,7-9E-BHVC in
the absence and in the presence of DNA or RNA with 100 : 1 [NA]/[dyes]
ratio. b: Two-photon action absorption spectra of 2,7-9E-BHVC in DNA
and RNA with 100 : 1 [NA]/[dyes] ratio. Compound concentration: 1.0 ¥
10-5 M.
maximum emission appears at about 600 nm. According to the
measurement data, in tris-HCl buffer, its U ¥ d is 1.67 GM, but
the values become 31.56 GM and 27.42 GM in DNA and RNA,
respectively (Table S1†), which show ca. 18-fold enhancement.
This demonstrated that 2,7-9E-BHVC can be used as a two-
photon fluorescence “light-switch” for NA. Moreover, taking into
account that the 2,7-9E-BHVC in NA solution has no linear
absorption in the spectral range of 700–900 nm and the maximum
absorption wavelength is at 450 nm, the two-photon action spectra
of 2,7-9E-BHVC in NA solution were determined by Ti:sapphire
laser sources tuneable at 750–840 nm (Fig. 2b). The maximum
U ¥ d of the compound in DNA and RNA solutions are about
44.22 GM and 48.89 GM at 830 nm. It is worth understanding
that 2,7-9E-BHVC possesses a larger U ¥ d in comparison with
commercial NA dyes, such as DAPI-DNA (2.18 GM)26 and EB-
DNA (0.91 GM).32 Therefore, one can reasonably expect that,
once the compound binds to NA particularly in an intracellular
environment, it will be a good two-photon fluorescent NA probe.
Fig. 3 shows the wide-field fluorescence microscopy image
of pretreated HeLa, SiHa and MS1 cells stained with 2,7-9E-
BHVC. The green fluorescence of 2,7-9E-BHVC mainly localizes
at the cytoplasm and nucleoli accompanied with faint nuclei
distribution in all three kinds of cells. Additionally, as shown
in Fig. 4, after treatment with ribonuclease A (RNase) which
only hydrolyzes the RNA and does not influence the DNA,
the fluorescence of 2,7-9E-BHVC in cytoplasm and nucleoli
dramatically diminished and tended to redistribute to the nucleoli
(Fig. 4c) in contrast to the untreated sample (Fig. 4a). However,
there was no apparent effect on the staining result of DAPI when
digested with RNase, because the DAPI tends to stain double-
stranded DNA rather than RNA. The digested results reconfirm
that 2,7-9E-BHVC prefers RNA to DNA in the complex internal
environment of cells, although there was no apparent disparity in
solution fluorescence measurements. A similar phenomenon was
also reported by Chang9 and Ohulchanskyy,25 but the mechanism
Fig. 1 a: The UV–vis absorption and single-photon excited fluorescence
spectra of 2,7-9E-BHVC in buffer (black), DNA (red) and RNA (green) at
100 : 1 [NA]/[dyes] ratio. b: Fluorometric titration curve of 2,7-9E-BHVC,
fluorescence intensity versus the [NA]/[dyes] ratio from 0 to 180 : 1.
Compound concentration: 2.0 ¥ 10-6 M.
Fig. 3 Wide-field fluorescence images of (a) HeLa, (b) SiHa and (c)
MS1cells stained with 5 mM 2,7-9E-BHVC for 30 min. Bar = 20 mm.
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different which may reflect the distinguishment of transcriptional
activity in different cell lines.
Fig. 6 shows the counterstain result of 2,7-9E-BHVC and
DAPI. The green fluorescence from nucleoli and cytoplasm is
easily discriminated from the nuclear zone with blue fluorescence,
and it means that 2,7-9E-BHVC has very good counterstain
compatibility with DAPI. The characteristics of 2,7-9E-BHVC
could help to image RNA distribution in relation to DNA in
cells and probably to reveal different patterns of RNA-DNA
colocalization which are cell type dependent.
Fig. 6 TPM fluorescence imaging of HeLa cells stained with 5 mM
2,7-9E-BHVC for 30 min followed by staining with 1 mM DAPI for
30 min. a: fluorescence of 2,7-9E-BHVC. b: fluorescence of DAPI. c:
merged picture of a and b. Bar = 20 mm.
Fig. 4 The RNase digest experiment. Wide-field fluorescence imaging
of cells stained with (a) 2,7-9E-BHVC and (b) DAPI. Cells stained
with (c) 2,7-9E-BHVC ◦and (d) DAPI followed by treatment with RNase
(30 mg ml-1) for 2 h at 37 C. Bar = 20 mm. Conditions: 5 mM, 2,7-9E-BHVC;
1 mM, DAPI; incubation time: 30 min.
Conclusions
In conclusion, we have developed a carbazole-centered dicationic
compound 2,7-9E-BHVC based on an A–p–D–p–A structure.
Very importantly, when binding to NA, 2,7-9E-BHVC possesses
bigger U ¥ d and notable two-photon fluorescence enhancement
in comparison with itself. At the same time, the experimental
results from wide-field fluorescence microscopy and TPM showed
it preferred RNA to DNA in cells. Moreover, it has good
counterstain compatibility with DAPI and is suitable for DNA-
RNA colocalization experiments.
has not been elucidated clearly. The possible reasons for this
puzzling state are two-fold: 1) some specificity of intracellular
distribution and organization of RNA and DNA molecules can
cause a difference in the affinity of 2,7-9E-BHVC to DNA and
RNA inside cells25 and 2) the structure of NA in solution may not
exactly reflect their real state in cells. Further investigations are
being executed to clarify this point.
The TPM imaging data of HeLa and SiHa cells stained with
2,7-9E-BHVC are shown in Fig. 5, and the position, shape and
amounts of the nucleoli labeled by 2,7-9E-BHVC are the same with
the most dense, dark-phase region of the nucleus in DIC images
which definitely represents nucleoli. From Fig. 5, one can also see
that HeLa cells have larger amounts of nucleoli than SiHa and the
distribution of the nucleoli in the two kinds of cells are significantly
Acknowledgements
For financial support, we thank the National Science Founda-
tion of China (50673053, 50173015, 30771091, 50990061 and
50721002), NSFC/RGC (50218001), Open Point of State Key
Laboratory of Supramolecular Structure and Materials and Inno-
vation Fund for the Post-Doctoral Program of Shandong Province
(201003076). W.-Y.W. also acknowledges financial support from
the University Grants Committee of HKSAR, China (Project No.
[AoE/P-03/08]) and Hong Kong Baptist University (FRG/08-
09/I-20).
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