Combination of Aptamer-Functionalized Quantum Dots and

Quantitative Bio-Science 32(1), 25~29(2013)
Combination of Aptamer-Functionalized Quantum Dots and
Electrophoretic Mobility Shift Assay for Specific Detection and
Quantitation of Proteins of Interest
Yea Seul Cho†, Eun Jeong Lee†, Seonmi Shin, Sang Soo Hah*
Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University,
Seoul 130-701, Korea
(Received April 25, 2013; Revised May 6, 2013; Accepted May 8, 2013)
ABSTRACT
Quantum dots (QDs) are semiconducting nanoparticles with interesting optical properties. The outstanding fluorescent
properties of QDs including photostability, high quantum yield, narrow emission bands, broad excitation spectra, and
accessibility to versatile functionalization are one of the key advantages for their sensing applications. In the present study,
we report QD-functionalized aptamers for specific detection and quantitation of proteins of interest, based on electrophoretic mobility shift assay (EMSA). Among the wide variety of potential biofunctional groups, we used the highly selective
interaction between aptamers and oligohistidine or human chorionic gonadotropin (hCG), and the results demonstrate that
the combination of the QD-functionalized aptamers and EMSA can be one of the best sensing options for protein quantitation with low limit of detection. In principle, multiplex detection for several proteins on a single gel can be achieved by
our method, leading to facilitation and simplification of the routinely used protein detection procedure.
Key words : Aptamer, Electrophoretic mobility shift assay, Protein quantitation, Quantum dot
Introduction
It is of great importance to investigate the interactions between proteins and nucleic acids in consideration that they mediate a wide range of biological processes within a cell and these
specific interactions are crucial for regulation of the biological
functions of DNA and RNA [1]. To detect protein-nucleic acid
interactions in vitro, electrophoretic mobility shift assay (EMSA,
also known as gel retardation assay), originally developed with
the aim of quantifying interactions between DNA and proteins
[2,3] and since then evolved to be suitable for different purposes including the detection and quantification of RNA-protein interactions, has been one of the most commonly used affinity electrophoresis techniques for the characterization of interacting systems, such as identification of nucleic acid-binding
proteins and of the respective consensus DNA or RNA sequ-
ences [1]. Under proper conditions, EMSA can be also used
for quantitative purposes, for example, determination of binding affinities, kinetics, and stoichiometry [1].
Traditional procedures for EMSA, however, usually use
large quantities of nucleic acids for ethidium bromide staining
or radioactively labeled nucleic acids for sensitivity purposes
[1], which have limited their effectiveness in ‘multiplexing’
(simultaneous detection of multiple signals) without complex
instrumentation and processing. Moreover, the procedures are
not economically preferable because lower concentrations of
nucleic acids are in general recommended in order to limit or
reduce nonspecific interactions in binding experiments [1].
Alternatively, nonradioactive systems, such as luminescent systems that reach similar performance without the disadvantages
of working with radioactivity, make use of fluorescence probes
for detection in-gel with the aid of an appropriate imaging system, but this method has not been very popular to date because
†
These authors contributed equally to this work.
* Correspondence should be addressed to Dr. Sang Soo Hah, Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130701, Korea. Tel: +82-2-961-2186, Fax: +82-2-966-3701, E-mail: [email protected]
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Quantitative Bio-Science Vol. 32, No. 1, 2013
26
expensive instruments are required and their sensitivity is not
yet comparable to that of radioactive probes [1]. In these regards, it is important to note that Wang and Reed very recently
demonstrated that aptamer-tethered gold nanoparticles could
be successfully used for direct visualization of protein-oligonucleotide interactions during gel electrophoresis [4].
On the other hands, the studies on the biological uses of semiconductor quantum dots (QDs) demonstrated that the unique
properties of QDs could overcome the issues and that the optical properties of QDs would be ideally suited for ‘multiplexing probes for EMSA [5-9]. The optical properties of QDs of
interest to biologists include high quantum yields, high molar
extinction coefficients (~10-100-fold higher than those of organic dyes), resistance to chemical degradation, photostability,
large and effective Stokes shifts, broad absorption with narrowsymmetric photoluminescence (PL) spectra (full-width at halfmaximum ~25-40 nm) spanning the UV to near-infrared, and
choice of size-tunable photoluminescence [5-9]. These properties also make them useful for multiplexing applications as
well as single-molecule tracking assays [5,6].
We previously reported the use of QD-conjugated RNA
aptamers targeting histidine tags (His-tags) as an alternative to
the conventional Western blot analysis [9]. Aptamers are a
special class of nucleic acids that can specifically bind, with
high affinity, to a target molecule [9-13]. Emerging as alternatives to antibodies, a wide range of aptamers have been found
to bind specifically to targets, thus they have been used in many
bioanalytical applications, such as for specific detection of proteins, metal ions, and small molecules, and for target-specific
delivery [9-13]. By virtue of the highly selective interaction of
an RNA aptamer complex with oligohistidine (Kd ~3.78 pM)
[9], which is comparable or superior to that of protein-antibody
(Kd ~10-5-10-12 M) [9], we could successfully develop a simple, time-saving, selective and sensitive method to detect the
His-tagged proteins. In the present study, we expand the scope
of the applicability of the RNA aptamer-functionalized QDs
to EMSA-based specific detection and quantitation of proteins
of interest.
Materials and Methods
1. General methods
Unless otherwise noted, reagents were obtained from commercial suppliers and were used without further purification,
and depc-treated deionized water was used for all experiments.
RNA aptamer-functionalized QDs were prepared as previously described [9]. In brief, QDs (emission maxima at 565 nm)
modified with PEG and amino groups were obtained from
Invitrogen (Carlsbad, CA). QD concentrations were measured
by optical absorbance, using extinction coefficients provided
by the supplier. Sulfosuccinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (sulfo-SMCC, Sigma) was used as
cross-linker. RNA-based aptamers for specific binding to Histagged proteins and human chorionic gonadotropin (hCG) were
synthesized, using the antisense oligonucleotide containing
the T7 promoter sequence at the 5′-end (5′-GCCAG CTCCC
GGGGC CAATC CCAAC CAGAC CACCC ATAGC CCCCC
CTATA GTGAG TCGTA TTAGT CC-3′) [6], and using the
antisense oligonucleotide containing the T7 promoter sequence
at the 5′-end (5′-TGAGC CTCCC GAAGG ATCGT TCAAC
GACCA ACGAG ACTCC CCCCT ATAGT GAGTC GTATT
AGTCC-3′) [14], respectively. The resulting RNA aptamer for
His-tagged protein or hCG was modified to contain a 5′-thiol
group, via an enzymatic method for the introduction of 5′-terminal sulfhydryl group at the 5′-termini of RNA molecules
according to the literature [9,15]. Prior to the transcription, the
5′-deoxy-5′-thioguanosine-5′-monophosphorothioate (GSMP)
was synthesized [15], as substrate for T7 RNA polymerase
that requires guanosine to efficiently initiate transcription. The
in vitro transcription followed by treatment of alkaline phosphatase was used to incorporate a sulfhydryl moiety to 5′-end
of RNA molecule. hCG was purchased from Calbiochem (Billerica, MA). All experiments were carried out at least in duplicate.
2. Preparation of E. coli containing His-tagged
proteins
His-tagged β-galactosidase (β-Gal-His) encoding plasmid
(pRSET/lacZ) was purchased from Invitrogen. Transformation
of E. coli BL21/DE3 harboring the pLysS vector was carried
out utilizing the transformation and storage solution method
[16]. Briefly, competent E. coli were prepared in LB broth
containing 15% polyethylene glycol. Afterwards, 0.2 μg of the
plasmid were added to 200 μL of competent cells and incubation was performed for 30 min at 0�
C. After incubation consecutively at 42�
C for 1.5 min and at 0�
C for 2 min, 0.8 mL of
LB medium were added followed by incubation at 37�
C for 1 h.
The cells were harvested by incubating the cells in LB broth
containing 50 mg/mL ampicilin and 35 mg/mL chloramphenicol. The cultivations were performed in shaking flasks with
100 mL of LB medium (10 g/L tryptone, 10 g/L NaCl, 5 g/L
Cho YS et al. : Aptamer and QD-based EMSA
Sulfo-SMCC
27
HS-RNA
QD
aptamer-functionalized QD
Scheme 1. Conjugation of anti-His-tag or anti-human chorionic gonadotropin (hCG) RNA aptamer on quantum dots (QDs).
3. EMSA-based quantitation of β-Gal-His and hCG
With the concentration of aptamer-conjugated QD fixed at
10 nM, different concentrations of β-Gal-His were mixed in
10 mM PBS buffer (pH 7.2) with 130 mM NaCl, 2.56 mM KCl,
and 9.5 mM NaHCO3, and incubated at 4�
C for 4 h. The resulting mixtures were subjected to hybrid (3% polyacrylamide and
0.5% agarose) gel electrophoresis according to the literature
[17], and the image was taken using a hand-held ultraviolet
light illuminator and a digital camera.
Results and Discussion
To accomplish the task to expand the scope of the applicability of the RNA aptamer-functionalized QDs to EMSA-based
specific detection and quantitation of proteins of interest, we
generated RNA aptamer-conjugated QDs, as illustrated in
Scheme 1.
After His-tagged proteins (β-Gal-His in this study) were
expressed in E. coli according to the literature [16], purified
β-Gal-His was quantified by the Bradford method. With the
(a)
β-Gal-His
aptamer-functionalized QD
bound to His-Tagged Protein
(b)
2.5
Migration distance (cm)
yeast extract) supplemented with 50 mg/mL ampicilin and 35
mg/mL chloramphenicol. At an optical density at 600 nm of
0.5-0.6, IPTG was added to induce the expression of β-GalHis, followed by 2-h incubation at 37�
C. The resulting cells
were harvested by centrifugation at 2,300×g for 10 min at 4�
C,
and the cell pellets were resuspended in 10 mM NaH2PO4,
140 mM NaCl, 2.7 mM KCl, pH 7.2, and disrupted at 0�
C with
ultrasound. After centrifugation and filtration the E. coli cell
lysates were used for further confirmation of the cellular Histagged protein expression. Confirmation of β-Gal-His from E.
coli cell lysate was performed utilizing Ni2+-loaded affinity
chromatography column (His SpinTrap, GE Healthcare) as
recommended by the manufacturer.
1.0
0
400
800
1200
[β-Gal-His] (nM)
Fig. 1. Combination of aptamer-functionalized QDs and electrophoretic mobility shift assay (EMSA). (a) Results of the combined assay
for detection and quantitation of His-tagged β-galactosidase (β-GalHis). Different concentrations of β-Gal-His were incubated with aptamer-functionalized QDs in 10 mM PBS buffer (pH 7.2) with 130 mM
C for
NaCl, 2.56 mM KCl, and 9.5 mM NaHCO3, and incubated at 4�
4 h and the resulting mixture was subjected to hybrid (3% polyacrylamide and 0.5% agarose) gel electrophoresis. Fluorescence image
demonstrating the achievement of 5 nM LOD (limit of detection) was
taken using a hand-held ultraviolet light illuminator and a digital
camera. (b) Plot of the migration distances as a function of β-Gal-His
concentration. Experiments were performed in triplicate. The data
may reveal apparent dissociation constant (Kdapp) between the QD conjugate and β-Gal-His under the employed conditions (~6.91 nM).
These data demonstrate that the combination of aptamer-functionalized QDs and EMSA can be an alternative to the conventional EMSA
and/or Western blot analysis for specific detection and quantitation of
proteins of interest.
concentration of aptamer-conjugated QD fixed at 10 nM, different concentrations of β-Gal-His were mixed and incubated
at 4�
C for 4 h. The resulting mixtures were subjected to hybrid
(3% polyacrylamide and 0.5% agarose) gel electrophoresis
28
Quantitative Bio-Science Vol. 32, No. 1, 2013
according to the literature [17], and the image was taken using
a hand-held ultraviolet light source and a digital camera to illuminate the bands and capture images of fluorescent bands. Fig.
1(a) shows that whereas some of the QD conjugates unbound
to β-Gal-His do not enter and separate well in the hybrid gel,
the fluorescent bands can result mainly from the QD conjugates bound to β-Gal-His and can be gradually retarded with
β-Gal-His concentrations increased. More importantly, the
data indicate that the migration distances can be plotted as a
function of β-Gal-His concentration (Fig. 1(b)). The typical
saturation curve obtained may reveal apparent dissociation
constant (Kdapp) between the QD conjugate and β-Gal-His
under the employed conditions. The inconsistency (approximately 2,000-fold difference) between the estimated value and
the reported Kd one [9] and the relatively big error bars in Fig.
1(b) are ascribed presumably to the negative effects from the
conjugation of the RNA aptamer onto the surface of QDs and
the electrophoresis conditions, and/or to the number of aptamers introduced onto the surface of each QD.
Furthermore, specific detection and quantitation of human
chorionic gonadotropin (hCG) was tested in a similar fashion.
As well known, hCG is a hormone produced during pregnancy
that is made by the developing placenta after conception, and
later by the placental component syncytiotrophoblast [18].
hCG can also be used as a tumor marker because some cancers including seminoma, choriocarcinoma, germ cell tumors,
hydatidiform mole formation secrete β subunit of hCG [18].
In general, hCG tests for pregnancy use antibody specific to
hCG, but antibodies have limited shelf life and are sensitive to
temperature and may undergo denaturation [18]. In this regard,
aptamers are good alternatives, since they can be produced by
chemical synthesis resulting in little or no batch-to-batch variation and are stable enough for long term storage [19]. Moreover, aptamers can be transported at ambient temperature and
denatured aptamers can be regenerated within minutes [19].
As shown in Fig. 2, different concentrations of hCG were
incubated with the anti-hCG RNA aptamer conjugated with
QDs, and the resulting mixture was subjected to hybrid gel
electrophoresis, in a similar manner as described. The fluorescence data show that the fluorescent bands can also result
mainly from the QD conjugates bound to hCG and can be
slowly retarded as the hCG concentration increases, suggesting that the method could be suitable for hCG detection and
quantitation. Our results collectively suggest that the combination of aptamer-functionalized QDs and EMSA can be alternatively used for specific detection and quantitation of pro-
hCG
0.1 μM
1 μM
5 μM
Fig. 2. Detection and quantitation of human chorionic gonadotropin
(hCG), and results of the combined assay for detection and quantitation of hCG. Binding experiments were performed in a similar fashion
as in Fig. 1. Experiments were carried out in duplicate.
teins, such as His-tagged proteins and hCG for pregnancy tests
or early cancer diagnostics.
Conclusion
To summarize, we employed QD-conjugated aptamer interactions with His-tag and hCG, leading to development of an
alternative to the conventional EMSA. Our assay is advantageous over the conventional radioactivity- or organic fluorophorebased EMSA due to the appreciated benefits of QDs and aptamers. Our present study brings the power of QD fluorescence
technology to a work-horse application in several biology-related fields, taking advantage of the unique properties of QDs and
aptamers as well as EMSA for specific detection and quantitation of proteins of interest. In principle, multiplex detection for
several proteins on a single gel can also be achieved by our
method, leading to facilitation and simplification of the routinely used protein detection procedure.
Acknowledgements
This work was supported by Basic Science Program through
the National Research Foundation (NRF) of Korea funded by
the Ministry of Education, Science and Technology (MEST)
(Nos. 2011-0021956 and 2012-001680).
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