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Poly(A)-targeting molecular beacons: Fluorescence resonance energy transfer-based in vitro quantitation and time-dependent imaging in live cells
Analytical Biochemistry 429 (2012) 92–98
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Poly(A)-targeting molecular beacons: Fluorescence resonance energy transfer-based in vitro quantitation and time-dependent imaging in live cells Min Young Kim, Jisu Kim, Sang Soo Hah ⇑ Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 14 June 2012 Received in revised form 4 July 2012 Accepted 6 July 2012 Available online 22 July 2012 Keywords: Molecular beacon mRNA Quantitative analysis Probing
a b s t r a c t Quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)+-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level using a fluorescence resonance energy transfer (FRET)-based molecular beacon are described. FRET-based molecular beacons were designed as poly(A)-targeting probes to be oligonucleotides that contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection (from 1.25 nM to 0.5 lM) of poly(A)-RNA, as well as for determination of Kd values, and revealed a distribution of the probe itself and localization of the target RNA sequence in cells. Furthermore, time-dependent FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed. Ó 2012 Elsevier Inc. All rights reserved.
Over the past decade, there has been increasing evidence to suggest that RNA molecules play important roles in many fundamental processes in cells with remarkable features, including regulation of protein biosynthesis, RNA splicing, retroviral replication, structural support for molecular machines, and gene silencing, of which the functions have been realized by control of their expression level and their spatial and temporal distribution [1]. From this point of view, it is very important to investigate the RNA expression level within a cell population as well as the spatial and temporal variation of RNA within live cells. Much of the knowledge about RNA dynamics and localization in live cells has come from either in situ hybridization in fixed cells or the biochemical fractionation of subcellular components [2,3]. However, these conventional methods provide only a static picture at the time of fixation or fractionation. On the contrary, real-time imaging methods for specific RNAs in live cells can provide dynamic information on RNA synthesis, processing, transport, and localization. Many powerful methods, including fluorescent proteins, labeled oligonucleotide probes, and aptamers, have been reported so far to visualize and track single messenger RNA (mRNA)1 molecules in real time, yielding new insights into the cell biology of mRNA [4,5]. In particular, several molecular beacons with different fluorogenic probes whose fluorescent properties change on sequence-specific ⇑ Corresponding author. Fax: +82 2 966 3701. E-mail address: [email protected] (S.S. Hah). Abbreviations used: mRNA, messenger RNA; FRET, fluorescence resonance energy transfer; MEM, minimum essential medium; PBS, phosphate-buffered saline; TIR, total internal reflection; smFRET, single-molecule FRET; CCD, charge-coupled device. 1
0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.07.010
hybridization have been successfully used [4,5] because the hybridization of the probes to the mRNA targets may either split apart or bring together a pair of interactive dyes attached to the probes, thereby leading to the fluorescence restoration of a quenched fluorophore or allowing for fluorescence resonance energy transfer (FRET) between a donor and an acceptor fluorophore residing on separate probes (Fig. 1). FRET is a vibrational and relaxational resonance energy transfer between the excited states of the fluorogenic dyes in which the excitation energy is transferred from a donor to an acceptor without the emission of a photon, with inverse sixth-power dependence on the distance between the two dyes [6–8]. The efficiency of energy transfer is high when the emission spectrum of the donor substantially overlaps with the absorption spectrum of the acceptor and when the distance between them is 20 to 100 Å [6]. At an optimal distance, emission from the donor is suppressed and emission from the acceptor is enhanced, leading to a shift in the color of the emitted light toward light of a longer wavelength [6]. Such energy transfer processes could provide us with significant information on the distance and interaction between two dyes, based on the intensities and wavelength shifts of the absorption and emission [6]. FRET, therefore, is now an important technique for investigation of a variety of biological phenomena that produce changes in molecular proximity. The aim of this study was to use a FRET-based molecular beacon for quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)+-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level.
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Fig.1. Poly(A)-targeting molecular beacons labeled with Cy3 at the 30 end and Cy5 at the 50 end as a FRET pair. Poly(A)-RNA binding to the probes causes conformational change, resulting in dissociation of the FRET pair. Thus, poly(A)-RNA can be quantified based on the emission change at the donor’s and/or acceptor’s characteristic wavelength.
Materials and methods Materials and reagents Reagents were obtained from commercial suppliers and were used without further purification unless otherwise noted. DNA hybridization probe (GCTCG TTTTT TTTTT TTTTT TTTTT TTTTT TAC GA GC) doubly conjugated with Cy5 and Cy3 at the 50 and 30 termini, respectively, and poly(A)-RNA (AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA) were synthesized and purified by Bioneer (Daejeon, Korea), and the probe concentration was determined by absorption at 260 nm with an Agilent 8453 spectrophotometer (Agilent). Diethylpyrocarbonate (DEPC)-treated deionized water was used throughout the experiments. Fluorescence spectra Fluorescence spectra of the probes (0.5 lM) annealed with poly(A)-RNA according to the literature were measured in a Trisbuffered solution (10 mM Tris–HCl and 4 mM magnesium chloride, pH 8.0) at room temperature. A FRET signal was obtained at 666 nm with an excitation at 552 nm, and spectra were recorded using a Synergy Mx spectrofluorophotometer (BioTek).
phenol red free serum-supplemented MEM. The cells were maintained under culture conditions using an incubation system during fluorescence imaging. Fluorescence images were acquired with a motorized inverted microscope (Axio Observer Z1, Zeiss). Acquired images were analyzed and processed with AxioVision software (version 4.8). The fluorescence and differential interference contrast (DIC) images were acquired sequentially within 2 s with a motorized microscope. Total internal reflection-based single-molecule FRET measurement Single-molecule fluorescence detection was performed by a total internal reflection (TIR)-based single-molecule FRET (smFRET) instrument [7]. Our system is constructed around a commercial inverted microscope (Zeiss) equipped with 532-nm DPSS laser system (Nd:YAG continuous crystal laser, 50 mW) and an electronmultiplying charge-coupled device (CCD) camera (iXon, Andor Technology). The fluorescence signals from Cy3 and Cy5 that were collected by a water immersion (prism-type) objective lens went through a longpass filter to block out laser scattering, and the signals lower and higher than 611 nm were separated by a dichroic mirror and detected by a CCD camera with up to 0.2 s time resolution. The observation area was 25 50 lm. Single-molecule fluorescence detection
Cell culture Culture reagents were purchased from Invitrogen. HeLa cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in minimum essential medium (MEM) containing 10% fetal bovine serum (FBS), 10 U/ml penicillin, and 0.01 mg/ml streptomycin. All cells were cultured at 37 °C under a humidified atmosphere of 5% CO2 in air, and exponentially growing cells were cultured following standard procedures until approximately 50% confluence was reached. Cells (50,000) were plated and harvested by means of trypsinization (1 ml of 0.05% trypsin solution for 1 min at 37 °C), and the resulting single-cell suspension was plated in 6-cm wells at 1 105 cells per well for 12 to 16 h prior to the doubly labeled probe as described below. Prior to microscope observation, the culture medium was washed and exchanged to phenol red free MEM.
Single-molecule fluorescence detection was carried out by adapting literature procedures [7]. For a chamber slide preparation, trypsinized HeLa cells were incubated at 37 °C with Lipofectamine 2000 and a hybridization probe (1 pmol) for transfection and deposition on the naked glass surface for 30 min. After washing the surface with PBS, the chamber slide was assembled with a coverslip. For the single-molecule fluorescence detection using the TIR-based smFRET instrument, the chamber slide was mounted on the inverted microscope as reported previously [7]. The resulting fluorescence signals were recorded in real time with a resolution of 0.2 s per scan using home-written Visual C++ software (Microsoft). The software was used to obtain each frame of the movie from the camera, and the single-molecule traces were extracted from the recorded movie file using scripts written in IDL (Research Systems). Results and discussion
Transfection and fluorescence imaging The doubly labeled probe (30 pmol) was introduced using 0.75 lg of Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. After 3 h of incubation at 37 °C with the Lipofectamine 2000 and a hybridization probe, the cells were washed three times with phosphate-buffered saline (PBS) and observed in
In vitro quantitation of poly(A)-RNA and determination of its dissociation constant with the poly(A)-targeting molecular beacon conjugated with FRET pair In the current study, we performed in vitro quantitation of poly(A)-RNA in an annealing buffer using oligonucleotides that
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contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. As illustrated in Fig. 1, this FRET-based molecular beacon assay relies on the probe with a labeled fluorescent donor at one end and an acceptor, either a fluorophore or a quencher, at the other end [4], enabling the molecule to assume a hairpin configuration where FRET donor and acceptor are held in close proximity and thereby allowing FRET between the donor and the acceptor to occur in the absence of the target. In the presence of the target, however, the formation of a probe–target hybrid disrupts the hairpin stem, removing the donor from the vicinity of the acceptor, restoring the donor’s fluorescence, and revealing the presence of the target [4], as clearly demonstrated in Fig. 2A. The target binding can be quantified based on the emission change at the donor’s and/or acceptor’s characteristic wavelength. Fig. 2 shows that the fluorescence intensities of the FRET acceptor, Cy5, are decreased as the amounts of poly(A)-RNA are increased, allowing for highly specific and wide-ranging detection (from 0.25 pmol to 1 nmol) of poly(A)-RNA. The linear range outlined in the inset of Fig. 2B demonstrates the achievement of a 1-nM limit of detection, which suggests that the described method may be applied to quantitation of poly(A)-RNA in total RNA samples. Analysis of ligand binding experiments may be based on a simple model, called the general law of mass action for the binding
interaction [9], and fractional occupancy or binding coefficient can be defined as the fraction of all receptors that are bound to poly(A)-RNA as described in Eq. (1), having allowed for estimation of apparent dissociation constant (Kdapp) of approximately 27.8 nM because fractional occupancy is 0.5 when [Ligand]0 = Kdapp (Fig. 2B):
Fractional Occupancy ¼
½Ligand0 ½Ligand0 þ K app d
ð1Þ
More intriguingly, we made use of the obtained results for Kd determination based on the fact that FRET signal is proportional to the amount of bound FRET pair because FRET-based technology can provide unique advantages over the traditional techniques for Kd determination, such as the surface plasmon resonance method and radiolabeled ligand binding assay, and because FRET technology allows real-time monitoring of the interactions of interest even in a multiple complex [10,11]. Furthermore, the concentrations in the system can be accurately determined by fluorescent emission intensity and quantum yield [12]. To implement this FRET-based technology for Kd determination, the fluorescence signals at 666 nm were differentiated into three fractions—FRET emission (EmFRET), Cy3 emission (Cy3(cont)), and Cy5 direct emission (Cy5(cont))—when excited at 552 nm (Eq. (2)), as shown in Fig. 3:
Fig.2. (A) Fluorescence spectra of poly(A)-targeting molecular beacons in the presence of poly(A)-RNA, showing that the beacon in a buffer produces a 10 to 50% FRET signal change in response to 1.25 nM to 0.5 lM poly(A)-RNA. (B) Fluorescence intensities of the Cy5 emission at 666 nm as a function of poly(A)-RNA concentration. The inset shows a logarithmic scale graph for the obtained data, demonstrating the achievement of a 1-nM limit of detection.
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Fig.3. Spectrum analysis of FRET signals showing dissection of the emission spectra from the molecular beacons labeled with Cy3 and Cy5 as a FRET pair. See the text for discussion.
EmFRET ¼ Emtotal Cy3ðcontÞ Cy5ðcontÞ ;
ð2Þ
where Cy3(cont) is the fluorescence signal contribution of the donor and Cy5(cont) is the fluorescence signal contribution of the acceptor. It was found out that the emission of the donor, Cy3, at 666 nm is proportional to its emission at 570 nm when excited at 552 nm with a ratio factor of x, whereas the direct emission of the acceptor, Cy5, at 666 nm is proportional to its emission at 666 nm when excited at 570 nm with a ratio factor of y (Eq. (3)):
EmFRET ¼ Emtotal x FLDD y FLAA ;
ð3Þ
where, FLDD is the fluorescence signal of the donor when excited at the donor wavelength (552 nm) and FLAA is the fluorescence signal of the acceptor when excited at the acceptor wavelength (570 nm). Measurement of different concentrations of Cy3 alone or Cy5 alone allowed us to determine x = 0.323 and y = 0.041. To determine the Kd between the FRET-based molecular beacon and poly(A)-RNA, we needed to convert the concentrations to functions of FRET signal intensity and to derive its relationship with Kd. Because the total concentration of the probe in our assay is fixed, the formula of Kd can be expressed as Eq. (4):
½probefree ½polyðAÞ-RNAfree ½probefree ½polyðAÞ-RNAfree ¼ ½probe polyðAÞ-RNA ½polyðAÞ-RNAbound f½probetotal ½polyðAÞ-RNAbound g ½polyðAÞ-RNAfree ¼ ½polyðAÞ-RNAbound
Kd ¼
¼
f½polyðAÞ-RNAmax bound ½polyðAÞ-RNAbound g ½polyðAÞ-RNAfree ½polyðAÞ-RNAbound ð4Þ
which can be converted to
½polyðAÞ-RNAbound ¼
½polyðAÞ-RNAmax bound ½polyðAÞ-RNAfree K d þ ½polyðAÞ-RNAfree
ð5Þ
Because the FRET signal intensity (EmFRET) is proportional to [poly(A)-RNA]bound in the mixture with all other parameters fixed, [poly(A)-RNA]bound in each condition is then determined by the sensitized emission intensity in a linear relationship (Eq. (6)):
½polyðAÞ-RNAbound ¼ ½polyðAÞ-RNAmax bound
EmFRET Emmax FRET
ð6Þ
Based on the general law of mass action for the binding interaction, the definition of Kd, and the combination of Eqs. (5) and (6), Kd can now be derived by fitting the dataset of EmFRET and total
poly(A)-RNA concentration in each condition. In our Kd determination experiment, the concentration of FRET-based molecular beacons was fixed to 0.01 lM and that of poly(A)-RNA was increased from 0 to 0.5 lM in a total volume of 200 ll. The fluorescence emission spectra of the mixture were then determined under an excitation wavelength of 552 nm, and after the FRET emission intensity under each condition was calculated, the dataset of EmFRET and [poly(A)-RNA]total were fitted by least-squares fitting, resulting in Kd being determined to be 15.1 ± 1.7 nM (Fig. 4), consistent with the literature [13]. This Kd value suggests that the FRET-based probes could compete with poly(A) binding proteins in live cells, as discussed below.
Time-dependent imaging of endogenous poly(A)+-RNAs with hybridization probes in HeLa cells Live cell imaging methods using fluorescent proteins and molecular beacons promise a finer temporal and spatial resolution of RNA dynamics and powerful new analytical possibilities [4,5]. Among the possibilities for selection, oligonucleotides labeled with only a single fluorophore have been used to image mRNAs in live cells [4]. For example, oligo(dT) and oligo(rU), labeled with a single fluorophore, have been used to bind to nuclear mRNAs and to study their mobility by fluorescence correlation spectroscopy or by fluorescence recovery after photobleaching [14,15]. However, these probes might not be suitable for live cell imaging because they inherently emit fluorescence even in the absence of their target. The distribution and intensity of probe fluorescence corresponds to the distribution and concentration of the probe itself, not always reflecting the distribution and concentration of its target RNA. For greater precision in RNA monitoring, observation of both the distribution of the probes and the localization of their target RNA is important [4]; thus, development of probes with properties allowing this simultaneous observation is required. The hybridization-sensitive fluorescent probes, such as traditional molecular beacons with both a fluorophore and a quencher, which possess the ability for fluorescence to be emitted only when they are hybridized with the target RNA, have been successfully used [4]. In this regard, we tested the hybridizationsensitive FRET-based molecular beacon to image the distribution and dynamics of poly(A)+-RNAs in live cells. Having established the fluorescence behavior of FRET-based molecular beacons in vitro, we examined the fluorescence imaging of human cancer cells, HeLa cells, where the probe was transfected.
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Fig.4. Kd determination of poly(A)-targeting molecular beacons for poly(A)-RNA. The concentration of FRET-based molecular beacons was fixed to 0.01 lM, and that of poly(A)-RNA was increased from 0 to 0.5 lM in a total volume of 200 ll. After the FRET emission intensity under each condition was calculated, the dataset of EmFRET and [poly(A)-RNA]total were fitted by least-squares fitting, resulting in Kd being determined to be 15.1 ± 1.7 nM.
The FRET-based molecular beacon (30 pmol) was transfected into HeLa cells by 3 h of incubation with Lipofectamine 2000, and then the fluorescence emission from the cells was examined using a green filter set (ex 500/24–25, dm 520, em 542/25–27) and a red filter set (ex 575–625, dm 645, em 660–710). As shown in Fig. 5 (see also Fig. S1 in the supplementary material), the fluorescence images from Cy3 and Cy5 of the probe in cells appeared to be different. Cy5 fluorescence, which indicates distribution of the probe as well as localization of poly(A)+-RNA through probe hybridization, was strong but time-dependently decreased in nuclei, whereas relatively weaker fluorescence was observed in the cytoplasmic area. Interestingly, the Cy3 fluorescence caused by FRET disappeared over 1 h, suggesting both that the probe would be resistant to hydrolysis by the nucleases in the cytosol and that the probe could compete with some poly(A) binding proteins in the cells, thereby resulting in very slow hybridization with to poly(A)+-RNA, as previously reported in the literature [16]. The fluorescence image obtained from the probe-transfected HeLa cells also explained the fluorescence property of the probe. Our probe, which can be hybridized with mRNAs that are normally polyadenylated in mammalian cells, seems to have only a small chance of binding with RNA in cells. When the probe was transfected into HeLa cells, intracellular uptake of the probe could be confirmed by the fluorescence of Cy3. The Cy3 fluorescence of the probe after washing was distributed mainly in the nuclei and was strong until 60 min of incubation. In contrast, the distribution of the Cy5 fluorescence of the probe was different from that of Cy3 fluorescence. Cy5 fluorescence arises from the hairpin structure prior to hybridization with the complementary RNA (i.e., the distribution of the fluorescence of Cy5 in cells indicates the localization of the complementary RNA) or to intracellular hydrolysis by nucleases. The observed Cy5 fluorescence, however, indicates that some of the probes are not a result of accumulation of the fluorescent probe but rather an accumulation of the poly(A)+-RNA hybridized with the probe. The fluorescence images with the Cy5 fluorescence showed both the distribution of the probe in the cells and the intracellular RNA localization. It should be noted, however, that the possibility of the probe being hydrolyzed by the nucleases in the cytosol cannot be excluded [4]. Imaging single poly(A)+-mRNA molecules Prompted by the results from the cell-based imaging experiment with the hybridization probe, we made use of a TIR-based
Fig.5. Time-dependent imaging of endogenous poly(A)+-mRNAs with poly(A)targeting molecular beacons in HeLa cells. The doubly labeled probe was introduced using Lipofectamine 2000 to HeLa cells for 3 h at 37 °C, followed by three times PBS washing. The cells were also maintained under normal culture conditions for the indicated time, and fluorescence images were acquired with a motorized inverted microscope. First column: phase contrast images; second column: fluorescence from Cy3 at 580 nm after 552 nm excitation; third column: FRET images due to the energy transfer from Cy3 to Cy5 fluorescence at 666 nm after 552 nm excitation; fourth column: merged images.
smFRET method, which is one of the recently developed techniques for probing individual molecules and watching relatively unperturbed molecular dynamics on the large scale of 2 to 8 nm [7,17]. Because this technique allows us to achieve relatively high throughput (200–400 molecules in an imaging area) if TIR-based smFRET data are taken with CCD cameras [17], we investigated the single-molecule spectroscopic events of the probe in live cells, which might provide us with valuable information on its time-resolved hybridization with poly(A)+-RNA in live cells. Although fluorescence recovery after photobleaching can be used to study the underlying dynamics [4], it only measures the average mobility of molecules in a defined subcellular zone and overlooks the variations and directionality in their motions. Therefore, single-molecule experiments that can directly visualize and track individual mRNA molecules are powerful tools in probing photophysical events because they reveal information hidden by ensemble averaging in bulk experiments, including static and dynamic heterogeneity, and because single-molecule sensitivity can allow for the detection of events too rare to perturb the ensemble-averaged signal [17]. We recently reported [7] that the TIR-based smFRET method could be used to investigate the single-molecule spectroscopic events of quantum dot (QD) fluorescence induced by NADPHdependent biocatalyzed transformation. This method has been employed in the current study for imaging single poly(A)+-RNA molecules. To study the fluorescence properties of the single-FRETbased molecular beacons resulting from the conformational changes on binding to the target poly(A) sequence, our system is constructed around a commercial inverted microscope equipped with a 532-nm DPSS laser system (Nd:YAG continuous crystal laser) and an electron-multiplying CCD camera. The fluorescence signals from Cy3 that were collected by a water immersion (prismtype) objective lens went through a longpass filter to block out laser light scattering. Signals lower and higher than 611 nm were separated by a dichroic mirror and detected by a CCD camera with up to 0.2 s time resolution. The observation area was 25 50 lm.
Poly(A)-targeting molecular beacons / M.Y. Kim et al. / Anal. Biochem. 429 (2012) 92–98
Taking advantage of the fixed locations of probe-transfected cells on the chamber slide, we tracked the fluorescence intensities of the single molecular beacons with a TIR-based smFRET instrument as a function of time (Fig. 6). Time trace data of the fluorescence emission from each fluorophore were recorded under continuous 532-nm laser excitation for 40 s with a resolution of 0.2 s per scan. For this TIR-based smFRET experiment, approximately 1 105 cells were incubated with 1 pmol of probe molecules as described, in consideration of the low transfection efficiency of the probe and the detectable Cy3 fluorescence caused by FRET. We could observe single-molecule spectroscopic events at this concentration of the probe, as clearly demonstrated in Fig. 6. The data show that the single-molecule spectroscopic events of the fluorescence changes of Cy3 and Cy5 in cells dramatically differed under constant laser excitation, with the patterns of the fluorescence increase of Cy3 and the fluorescence decrease of Cy5 reversely and significantly correlated for 40 s, and that the fluorescence of Cy5 almost disappeared within 30 min, whereas that of Cy3 was slightly intensified, as could be typically observed from the TIR-based smFRET experiments. Taken together, our observations strongly suggest that the time-dependent FRET-mediated fluorescence changes at the single-molecule level could be caused
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by the folding-induced conformation changes of the probe in live cells. This result also suggests that it would be interesting to apply this technology to investigate the time-resolved dynamics of poly(A)+-RNA processing under both physiological and diseased conditions in the future so as to understand the cellular pathology of human diseases. Because single-molecule tracking can reveal the range of behaviors of the individual molecules and define characteristics of the cellular matrix in which they travel, and because only a few copies of most mRNAs are usually expressed in each cell, methods for the detection of individual mRNA molecules using FRET-based molecular beacons targeting specific translated regions in mRNA are expected to provide the only means of seeing where these RNAs go and how they get there. To study the dynamics of mRNA synthesis and degradation in more detail, however, it is desirable to use probes that respond rapidly to the appearance of target mRNA and are then degraded or turned off when the target mRNA is degraded. Conclusion In this work, we have reported quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)+-RNA locali-
Fig.6. Single-molecule images of HeLa cells transfected with poly(A)-targeting molecular beacons. For a chamber slide preparation, trypsinized HeLa cells were incubated at 37 °C with Lipofectamine 2000 and the probe for transfection and deposition on the naked glass surface for 30 min. After washing the surface with PBS, the chamber slide was assembled with a coverslip. For the single-molecule fluorescence detection using the TIR-based smFRET instrument, the chamber slide was mounted on the inverted microscope after additional incubation for the indicated time under normal culture conditions. The resulting fluorescence signals were recorded in real time with a resolution of 0.2 s per scan for 40 s. The image is split into Cy3 (<611 nm) and Cy5 (>611 nm) emission channels, each 25 50 mm. Below are time trace data of the fluorescence emission from each fluorophore, demonstrating that the single-molecule spectroscopic events of the fluorescence changes of Cy3 and Cy5 in cells clearly differ under constant laser excitation and that the fluorescence of Cy5 almost disappears within 30 min, whereas that of Cy3 is slightly intensified.
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zation in living mammalian cells, and time-resolved intracellular binding dynamics of FRET-based molecular beacons at the singlemolecule level. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection of poly(A)-RNA and revealed a distribution of the probe itself and localization of the target RNA sequence in live cells. Moreover, FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed, for which the method is expected to reveal information hidden by ensemble averaging in bulk experiments and to allow for the detection of events too rare to perturb the ensemble-averaged signal. Acknowledgments We are very grateful to Young Dong Kim (Kyung Hee University) for his kind help. This work was supported by the Basic Science Program through the National Research Foundation of Korea (KRF) funded by the Ministry of Education, Science, and Technology (MEST) (2011-0021956 and 2012-001680). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2012.07.010. References [1] G.F. Joyce, The antiquity of RNA-based evolution, Nature 418 (2002) 214–216. [2] M.P.C. van de Corput, R.W. Dirks, R.P.M. van Gijlswijk, E. van Binnendijk, C.M. Hattinger, R.A. de Paus, J.E. Landegent, A.K. Raap, Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxide-labeled oligonucleotides and tyramide signal amplification, J. Histochem. Cytochem. 46 (1998) 1249–1259.
[3] A.M. Femino, F.S. Fay, K. Fogarty, R.H. Singer, Visualization of single RNA transcripts in situ, Science 280 (1998) 585–590. [4] S. Tyagi, Imaging intracellular RNA distribution and dynamics in living cells, Nat. Methods 6 (2009) 331–338. [5] G. Bao, W.J. Rhee, A. Tsourkas, Fluorescence probes for live-cell RNA detection, Annu. Rev. Biomed. Eng. 11 (2009) 25–47. [6] B. Valeur, Molecular Fluorescence. Principles and Applications, Wiley–VCH, Weinheim, Germany, 2002. [7] Y.S. Kim, M.Y. Kim, J.K. Song, T.J. Kim, Y.D. Kim, S.S. Hah, Stepwise fluorescence changes of quantum dots: single-molecule spectroscopic studies on the properties of turn-on quantum dots, Chem. Commun. 48 (2012) 723–725. [8] S. Shin, H.M. Kwon, K.S. Yoon, D.E. Kim, S.S. Hah, FRET-based probing to gain direct information on siRNA sustainability in live cells: asymmetric degradation of siRNA strands, Mol. Biosyst. 7 (2011) 2110–2113. [9] J. Kim, M.Y. Kim, H.S. Kim, S.S. Hah, Binding of uranyl ion by a DNA aptamer attached to a solid support, Bioorg. Med. Chem. Lett. 21 (2011) 4020–4022. [10] I.T. Li, E. Pham, K. Truong, Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics, Biotechnol. Lett. 28 (2006) 1971–1982. [11] K.E. Sapsford, L. Berti, I.L. Medintz, Materials for fluorescence resonance energy transfer analysis: beyond traditional donor–acceptor combinations, Angew. Chem. Int. Ed. Engl. 45 (2006) 4562–4589. [12] S.F. Martin, M.H. Tatham, R.T. Hay, I.D.W. Samuel, Quantitative analysis of multi-protein interactions using FRET: application to the SUMO pathway, Protein Sci. 17 (2008) 777–784. [13] V. Vijayanathan, T. Thomas, L.H. Sigal, T.J. Thomas, Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon, Antisense Nucleic Acid Drug Dev. 12 (2002) 225–233. [14] J.C. Politz, E.S. Browne, D.E. Wolf, T. Pederson, Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells, Proc. Natl. Acad. Sci. USA 95 (1998) 6043–6048. [15] C. Molenaar, A. Abdulle, A. Gena, H.J. Tanke, R.W. Dirks, Poly(A)+ RNAs roam the cell nucleus and pass through speckle domains in transcriptionally active and inactive cells, J. Cell Biol. 165 (2004) 191–202. [16] A. Calapez, H.M. Pereira, A. Calado, J. Graga, J. Rino, C. Carvalho, J.P. Tavanez, E. Wahle, A.C. Rosa, M. Carmo-Fonseca, The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive, J. Cell Biol. 159 (2002) 795–805. [17] R. Roy, S. Hohng, T. Ha, A practical guide to single-molecule FRET, Nat. Methods 5 (2008) 507–516.
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Poly(A)-targeting molecular beacons: Fluorescence resonance energy transfer-based in vitro quantitation and time-dependent imaging in live cells Min Young Kim, Jisu Kim, Sang Soo Hah ⇑ Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 14 June 2012 Received in revised form 4 July 2012 Accepted 6 July 2012 Available online 22 July 2012 Keywords: Molecular beacon mRNA Quantitative analysis Probing
a b s t r a c t Quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)+-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level using a fluorescence resonance energy transfer (FRET)-based molecular beacon are described. FRET-based molecular beacons were designed as poly(A)-targeting probes to be oligonucleotides that contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection (from 1.25 nM to 0.5 lM) of poly(A)-RNA, as well as for determination of Kd values, and revealed a distribution of the probe itself and localization of the target RNA sequence in cells. Furthermore, time-dependent FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed. Ó 2012 Elsevier Inc. All rights reserved.
Over the past decade, there has been increasing evidence to suggest that RNA molecules play important roles in many fundamental processes in cells with remarkable features, including regulation of protein biosynthesis, RNA splicing, retroviral replication, structural support for molecular machines, and gene silencing, of which the functions have been realized by control of their expression level and their spatial and temporal distribution [1]. From this point of view, it is very important to investigate the RNA expression level within a cell population as well as the spatial and temporal variation of RNA within live cells. Much of the knowledge about RNA dynamics and localization in live cells has come from either in situ hybridization in fixed cells or the biochemical fractionation of subcellular components [2,3]. However, these conventional methods provide only a static picture at the time of fixation or fractionation. On the contrary, real-time imaging methods for specific RNAs in live cells can provide dynamic information on RNA synthesis, processing, transport, and localization. Many powerful methods, including fluorescent proteins, labeled oligonucleotide probes, and aptamers, have been reported so far to visualize and track single messenger RNA (mRNA)1 molecules in real time, yielding new insights into the cell biology of mRNA [4,5]. In particular, several molecular beacons with different fluorogenic probes whose fluorescent properties change on sequence-specific ⇑ Corresponding author. Fax: +82 2 966 3701. E-mail address: [email protected] (S.S. Hah). Abbreviations used: mRNA, messenger RNA; FRET, fluorescence resonance energy transfer; MEM, minimum essential medium; PBS, phosphate-buffered saline; TIR, total internal reflection; smFRET, single-molecule FRET; CCD, charge-coupled device. 1
0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.07.010
hybridization have been successfully used [4,5] because the hybridization of the probes to the mRNA targets may either split apart or bring together a pair of interactive dyes attached to the probes, thereby leading to the fluorescence restoration of a quenched fluorophore or allowing for fluorescence resonance energy transfer (FRET) between a donor and an acceptor fluorophore residing on separate probes (Fig. 1). FRET is a vibrational and relaxational resonance energy transfer between the excited states of the fluorogenic dyes in which the excitation energy is transferred from a donor to an acceptor without the emission of a photon, with inverse sixth-power dependence on the distance between the two dyes [6–8]. The efficiency of energy transfer is high when the emission spectrum of the donor substantially overlaps with the absorption spectrum of the acceptor and when the distance between them is 20 to 100 Å [6]. At an optimal distance, emission from the donor is suppressed and emission from the acceptor is enhanced, leading to a shift in the color of the emitted light toward light of a longer wavelength [6]. Such energy transfer processes could provide us with significant information on the distance and interaction between two dyes, based on the intensities and wavelength shifts of the absorption and emission [6]. FRET, therefore, is now an important technique for investigation of a variety of biological phenomena that produce changes in molecular proximity. The aim of this study was to use a FRET-based molecular beacon for quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)+-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level.
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Fig.1. Poly(A)-targeting molecular beacons labeled with Cy3 at the 30 end and Cy5 at the 50 end as a FRET pair. Poly(A)-RNA binding to the probes causes conformational change, resulting in dissociation of the FRET pair. Thus, poly(A)-RNA can be quantified based on the emission change at the donor’s and/or acceptor’s characteristic wavelength.
Materials and methods Materials and reagents Reagents were obtained from commercial suppliers and were used without further purification unless otherwise noted. DNA hybridization probe (GCTCG TTTTT TTTTT TTTTT TTTTT TTTTT TAC GA GC) doubly conjugated with Cy5 and Cy3 at the 50 and 30 termini, respectively, and poly(A)-RNA (AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA) were synthesized and purified by Bioneer (Daejeon, Korea), and the probe concentration was determined by absorption at 260 nm with an Agilent 8453 spectrophotometer (Agilent). Diethylpyrocarbonate (DEPC)-treated deionized water was used throughout the experiments. Fluorescence spectra Fluorescence spectra of the probes (0.5 lM) annealed with poly(A)-RNA according to the literature were measured in a Trisbuffered solution (10 mM Tris–HCl and 4 mM magnesium chloride, pH 8.0) at room temperature. A FRET signal was obtained at 666 nm with an excitation at 552 nm, and spectra were recorded using a Synergy Mx spectrofluorophotometer (BioTek).
phenol red free serum-supplemented MEM. The cells were maintained under culture conditions using an incubation system during fluorescence imaging. Fluorescence images were acquired with a motorized inverted microscope (Axio Observer Z1, Zeiss). Acquired images were analyzed and processed with AxioVision software (version 4.8). The fluorescence and differential interference contrast (DIC) images were acquired sequentially within 2 s with a motorized microscope. Total internal reflection-based single-molecule FRET measurement Single-molecule fluorescence detection was performed by a total internal reflection (TIR)-based single-molecule FRET (smFRET) instrument [7]. Our system is constructed around a commercial inverted microscope (Zeiss) equipped with 532-nm DPSS laser system (Nd:YAG continuous crystal laser, 50 mW) and an electronmultiplying charge-coupled device (CCD) camera (iXon, Andor Technology). The fluorescence signals from Cy3 and Cy5 that were collected by a water immersion (prism-type) objective lens went through a longpass filter to block out laser scattering, and the signals lower and higher than 611 nm were separated by a dichroic mirror and detected by a CCD camera with up to 0.2 s time resolution. The observation area was 25 50 lm. Single-molecule fluorescence detection
Cell culture Culture reagents were purchased from Invitrogen. HeLa cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in minimum essential medium (MEM) containing 10% fetal bovine serum (FBS), 10 U/ml penicillin, and 0.01 mg/ml streptomycin. All cells were cultured at 37 °C under a humidified atmosphere of 5% CO2 in air, and exponentially growing cells were cultured following standard procedures until approximately 50% confluence was reached. Cells (50,000) were plated and harvested by means of trypsinization (1 ml of 0.05% trypsin solution for 1 min at 37 °C), and the resulting single-cell suspension was plated in 6-cm wells at 1 105 cells per well for 12 to 16 h prior to the doubly labeled probe as described below. Prior to microscope observation, the culture medium was washed and exchanged to phenol red free MEM.
Single-molecule fluorescence detection was carried out by adapting literature procedures [7]. For a chamber slide preparation, trypsinized HeLa cells were incubated at 37 °C with Lipofectamine 2000 and a hybridization probe (1 pmol) for transfection and deposition on the naked glass surface for 30 min. After washing the surface with PBS, the chamber slide was assembled with a coverslip. For the single-molecule fluorescence detection using the TIR-based smFRET instrument, the chamber slide was mounted on the inverted microscope as reported previously [7]. The resulting fluorescence signals were recorded in real time with a resolution of 0.2 s per scan using home-written Visual C++ software (Microsoft). The software was used to obtain each frame of the movie from the camera, and the single-molecule traces were extracted from the recorded movie file using scripts written in IDL (Research Systems). Results and discussion
Transfection and fluorescence imaging The doubly labeled probe (30 pmol) was introduced using 0.75 lg of Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. After 3 h of incubation at 37 °C with the Lipofectamine 2000 and a hybridization probe, the cells were washed three times with phosphate-buffered saline (PBS) and observed in
In vitro quantitation of poly(A)-RNA and determination of its dissociation constant with the poly(A)-targeting molecular beacon conjugated with FRET pair In the current study, we performed in vitro quantitation of poly(A)-RNA in an annealing buffer using oligonucleotides that
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contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. As illustrated in Fig. 1, this FRET-based molecular beacon assay relies on the probe with a labeled fluorescent donor at one end and an acceptor, either a fluorophore or a quencher, at the other end [4], enabling the molecule to assume a hairpin configuration where FRET donor and acceptor are held in close proximity and thereby allowing FRET between the donor and the acceptor to occur in the absence of the target. In the presence of the target, however, the formation of a probe–target hybrid disrupts the hairpin stem, removing the donor from the vicinity of the acceptor, restoring the donor’s fluorescence, and revealing the presence of the target [4], as clearly demonstrated in Fig. 2A. The target binding can be quantified based on the emission change at the donor’s and/or acceptor’s characteristic wavelength. Fig. 2 shows that the fluorescence intensities of the FRET acceptor, Cy5, are decreased as the amounts of poly(A)-RNA are increased, allowing for highly specific and wide-ranging detection (from 0.25 pmol to 1 nmol) of poly(A)-RNA. The linear range outlined in the inset of Fig. 2B demonstrates the achievement of a 1-nM limit of detection, which suggests that the described method may be applied to quantitation of poly(A)-RNA in total RNA samples. Analysis of ligand binding experiments may be based on a simple model, called the general law of mass action for the binding
interaction [9], and fractional occupancy or binding coefficient can be defined as the fraction of all receptors that are bound to poly(A)-RNA as described in Eq. (1), having allowed for estimation of apparent dissociation constant (Kdapp) of approximately 27.8 nM because fractional occupancy is 0.5 when [Ligand]0 = Kdapp (Fig. 2B):
Fractional Occupancy ¼
½Ligand0 ½Ligand0 þ K app d
ð1Þ
More intriguingly, we made use of the obtained results for Kd determination based on the fact that FRET signal is proportional to the amount of bound FRET pair because FRET-based technology can provide unique advantages over the traditional techniques for Kd determination, such as the surface plasmon resonance method and radiolabeled ligand binding assay, and because FRET technology allows real-time monitoring of the interactions of interest even in a multiple complex [10,11]. Furthermore, the concentrations in the system can be accurately determined by fluorescent emission intensity and quantum yield [12]. To implement this FRET-based technology for Kd determination, the fluorescence signals at 666 nm were differentiated into three fractions—FRET emission (EmFRET), Cy3 emission (Cy3(cont)), and Cy5 direct emission (Cy5(cont))—when excited at 552 nm (Eq. (2)), as shown in Fig. 3:
Fig.2. (A) Fluorescence spectra of poly(A)-targeting molecular beacons in the presence of poly(A)-RNA, showing that the beacon in a buffer produces a 10 to 50% FRET signal change in response to 1.25 nM to 0.5 lM poly(A)-RNA. (B) Fluorescence intensities of the Cy5 emission at 666 nm as a function of poly(A)-RNA concentration. The inset shows a logarithmic scale graph for the obtained data, demonstrating the achievement of a 1-nM limit of detection.
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Fig.3. Spectrum analysis of FRET signals showing dissection of the emission spectra from the molecular beacons labeled with Cy3 and Cy5 as a FRET pair. See the text for discussion.
EmFRET ¼ Emtotal Cy3ðcontÞ Cy5ðcontÞ ;
ð2Þ
where Cy3(cont) is the fluorescence signal contribution of the donor and Cy5(cont) is the fluorescence signal contribution of the acceptor. It was found out that the emission of the donor, Cy3, at 666 nm is proportional to its emission at 570 nm when excited at 552 nm with a ratio factor of x, whereas the direct emission of the acceptor, Cy5, at 666 nm is proportional to its emission at 666 nm when excited at 570 nm with a ratio factor of y (Eq. (3)):
EmFRET ¼ Emtotal x FLDD y FLAA ;
ð3Þ
where, FLDD is the fluorescence signal of the donor when excited at the donor wavelength (552 nm) and FLAA is the fluorescence signal of the acceptor when excited at the acceptor wavelength (570 nm). Measurement of different concentrations of Cy3 alone or Cy5 alone allowed us to determine x = 0.323 and y = 0.041. To determine the Kd between the FRET-based molecular beacon and poly(A)-RNA, we needed to convert the concentrations to functions of FRET signal intensity and to derive its relationship with Kd. Because the total concentration of the probe in our assay is fixed, the formula of Kd can be expressed as Eq. (4):
½probefree ½polyðAÞ-RNAfree ½probefree ½polyðAÞ-RNAfree ¼ ½probe polyðAÞ-RNA ½polyðAÞ-RNAbound f½probetotal ½polyðAÞ-RNAbound g ½polyðAÞ-RNAfree ¼ ½polyðAÞ-RNAbound
Kd ¼
¼
f½polyðAÞ-RNAmax bound ½polyðAÞ-RNAbound g ½polyðAÞ-RNAfree ½polyðAÞ-RNAbound ð4Þ
which can be converted to
½polyðAÞ-RNAbound ¼
½polyðAÞ-RNAmax bound ½polyðAÞ-RNAfree K d þ ½polyðAÞ-RNAfree
ð5Þ
Because the FRET signal intensity (EmFRET) is proportional to [poly(A)-RNA]bound in the mixture with all other parameters fixed, [poly(A)-RNA]bound in each condition is then determined by the sensitized emission intensity in a linear relationship (Eq. (6)):
½polyðAÞ-RNAbound ¼ ½polyðAÞ-RNAmax bound
EmFRET Emmax FRET
ð6Þ
Based on the general law of mass action for the binding interaction, the definition of Kd, and the combination of Eqs. (5) and (6), Kd can now be derived by fitting the dataset of EmFRET and total
poly(A)-RNA concentration in each condition. In our Kd determination experiment, the concentration of FRET-based molecular beacons was fixed to 0.01 lM and that of poly(A)-RNA was increased from 0 to 0.5 lM in a total volume of 200 ll. The fluorescence emission spectra of the mixture were then determined under an excitation wavelength of 552 nm, and after the FRET emission intensity under each condition was calculated, the dataset of EmFRET and [poly(A)-RNA]total were fitted by least-squares fitting, resulting in Kd being determined to be 15.1 ± 1.7 nM (Fig. 4), consistent with the literature [13]. This Kd value suggests that the FRET-based probes could compete with poly(A) binding proteins in live cells, as discussed below.
Time-dependent imaging of endogenous poly(A)+-RNAs with hybridization probes in HeLa cells Live cell imaging methods using fluorescent proteins and molecular beacons promise a finer temporal and spatial resolution of RNA dynamics and powerful new analytical possibilities [4,5]. Among the possibilities for selection, oligonucleotides labeled with only a single fluorophore have been used to image mRNAs in live cells [4]. For example, oligo(dT) and oligo(rU), labeled with a single fluorophore, have been used to bind to nuclear mRNAs and to study their mobility by fluorescence correlation spectroscopy or by fluorescence recovery after photobleaching [14,15]. However, these probes might not be suitable for live cell imaging because they inherently emit fluorescence even in the absence of their target. The distribution and intensity of probe fluorescence corresponds to the distribution and concentration of the probe itself, not always reflecting the distribution and concentration of its target RNA. For greater precision in RNA monitoring, observation of both the distribution of the probes and the localization of their target RNA is important [4]; thus, development of probes with properties allowing this simultaneous observation is required. The hybridization-sensitive fluorescent probes, such as traditional molecular beacons with both a fluorophore and a quencher, which possess the ability for fluorescence to be emitted only when they are hybridized with the target RNA, have been successfully used [4]. In this regard, we tested the hybridizationsensitive FRET-based molecular beacon to image the distribution and dynamics of poly(A)+-RNAs in live cells. Having established the fluorescence behavior of FRET-based molecular beacons in vitro, we examined the fluorescence imaging of human cancer cells, HeLa cells, where the probe was transfected.
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Fig.4. Kd determination of poly(A)-targeting molecular beacons for poly(A)-RNA. The concentration of FRET-based molecular beacons was fixed to 0.01 lM, and that of poly(A)-RNA was increased from 0 to 0.5 lM in a total volume of 200 ll. After the FRET emission intensity under each condition was calculated, the dataset of EmFRET and [poly(A)-RNA]total were fitted by least-squares fitting, resulting in Kd being determined to be 15.1 ± 1.7 nM.
The FRET-based molecular beacon (30 pmol) was transfected into HeLa cells by 3 h of incubation with Lipofectamine 2000, and then the fluorescence emission from the cells was examined using a green filter set (ex 500/24–25, dm 520, em 542/25–27) and a red filter set (ex 575–625, dm 645, em 660–710). As shown in Fig. 5 (see also Fig. S1 in the supplementary material), the fluorescence images from Cy3 and Cy5 of the probe in cells appeared to be different. Cy5 fluorescence, which indicates distribution of the probe as well as localization of poly(A)+-RNA through probe hybridization, was strong but time-dependently decreased in nuclei, whereas relatively weaker fluorescence was observed in the cytoplasmic area. Interestingly, the Cy3 fluorescence caused by FRET disappeared over 1 h, suggesting both that the probe would be resistant to hydrolysis by the nucleases in the cytosol and that the probe could compete with some poly(A) binding proteins in the cells, thereby resulting in very slow hybridization with to poly(A)+-RNA, as previously reported in the literature [16]. The fluorescence image obtained from the probe-transfected HeLa cells also explained the fluorescence property of the probe. Our probe, which can be hybridized with mRNAs that are normally polyadenylated in mammalian cells, seems to have only a small chance of binding with RNA in cells. When the probe was transfected into HeLa cells, intracellular uptake of the probe could be confirmed by the fluorescence of Cy3. The Cy3 fluorescence of the probe after washing was distributed mainly in the nuclei and was strong until 60 min of incubation. In contrast, the distribution of the Cy5 fluorescence of the probe was different from that of Cy3 fluorescence. Cy5 fluorescence arises from the hairpin structure prior to hybridization with the complementary RNA (i.e., the distribution of the fluorescence of Cy5 in cells indicates the localization of the complementary RNA) or to intracellular hydrolysis by nucleases. The observed Cy5 fluorescence, however, indicates that some of the probes are not a result of accumulation of the fluorescent probe but rather an accumulation of the poly(A)+-RNA hybridized with the probe. The fluorescence images with the Cy5 fluorescence showed both the distribution of the probe in the cells and the intracellular RNA localization. It should be noted, however, that the possibility of the probe being hydrolyzed by the nucleases in the cytosol cannot be excluded [4]. Imaging single poly(A)+-mRNA molecules Prompted by the results from the cell-based imaging experiment with the hybridization probe, we made use of a TIR-based
Fig.5. Time-dependent imaging of endogenous poly(A)+-mRNAs with poly(A)targeting molecular beacons in HeLa cells. The doubly labeled probe was introduced using Lipofectamine 2000 to HeLa cells for 3 h at 37 °C, followed by three times PBS washing. The cells were also maintained under normal culture conditions for the indicated time, and fluorescence images were acquired with a motorized inverted microscope. First column: phase contrast images; second column: fluorescence from Cy3 at 580 nm after 552 nm excitation; third column: FRET images due to the energy transfer from Cy3 to Cy5 fluorescence at 666 nm after 552 nm excitation; fourth column: merged images.
smFRET method, which is one of the recently developed techniques for probing individual molecules and watching relatively unperturbed molecular dynamics on the large scale of 2 to 8 nm [7,17]. Because this technique allows us to achieve relatively high throughput (200–400 molecules in an imaging area) if TIR-based smFRET data are taken with CCD cameras [17], we investigated the single-molecule spectroscopic events of the probe in live cells, which might provide us with valuable information on its time-resolved hybridization with poly(A)+-RNA in live cells. Although fluorescence recovery after photobleaching can be used to study the underlying dynamics [4], it only measures the average mobility of molecules in a defined subcellular zone and overlooks the variations and directionality in their motions. Therefore, single-molecule experiments that can directly visualize and track individual mRNA molecules are powerful tools in probing photophysical events because they reveal information hidden by ensemble averaging in bulk experiments, including static and dynamic heterogeneity, and because single-molecule sensitivity can allow for the detection of events too rare to perturb the ensemble-averaged signal [17]. We recently reported [7] that the TIR-based smFRET method could be used to investigate the single-molecule spectroscopic events of quantum dot (QD) fluorescence induced by NADPHdependent biocatalyzed transformation. This method has been employed in the current study for imaging single poly(A)+-RNA molecules. To study the fluorescence properties of the single-FRETbased molecular beacons resulting from the conformational changes on binding to the target poly(A) sequence, our system is constructed around a commercial inverted microscope equipped with a 532-nm DPSS laser system (Nd:YAG continuous crystal laser) and an electron-multiplying CCD camera. The fluorescence signals from Cy3 that were collected by a water immersion (prismtype) objective lens went through a longpass filter to block out laser light scattering. Signals lower and higher than 611 nm were separated by a dichroic mirror and detected by a CCD camera with up to 0.2 s time resolution. The observation area was 25 50 lm.
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Taking advantage of the fixed locations of probe-transfected cells on the chamber slide, we tracked the fluorescence intensities of the single molecular beacons with a TIR-based smFRET instrument as a function of time (Fig. 6). Time trace data of the fluorescence emission from each fluorophore were recorded under continuous 532-nm laser excitation for 40 s with a resolution of 0.2 s per scan. For this TIR-based smFRET experiment, approximately 1 105 cells were incubated with 1 pmol of probe molecules as described, in consideration of the low transfection efficiency of the probe and the detectable Cy3 fluorescence caused by FRET. We could observe single-molecule spectroscopic events at this concentration of the probe, as clearly demonstrated in Fig. 6. The data show that the single-molecule spectroscopic events of the fluorescence changes of Cy3 and Cy5 in cells dramatically differed under constant laser excitation, with the patterns of the fluorescence increase of Cy3 and the fluorescence decrease of Cy5 reversely and significantly correlated for 40 s, and that the fluorescence of Cy5 almost disappeared within 30 min, whereas that of Cy3 was slightly intensified, as could be typically observed from the TIR-based smFRET experiments. Taken together, our observations strongly suggest that the time-dependent FRET-mediated fluorescence changes at the single-molecule level could be caused
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by the folding-induced conformation changes of the probe in live cells. This result also suggests that it would be interesting to apply this technology to investigate the time-resolved dynamics of poly(A)+-RNA processing under both physiological and diseased conditions in the future so as to understand the cellular pathology of human diseases. Because single-molecule tracking can reveal the range of behaviors of the individual molecules and define characteristics of the cellular matrix in which they travel, and because only a few copies of most mRNAs are usually expressed in each cell, methods for the detection of individual mRNA molecules using FRET-based molecular beacons targeting specific translated regions in mRNA are expected to provide the only means of seeing where these RNAs go and how they get there. To study the dynamics of mRNA synthesis and degradation in more detail, however, it is desirable to use probes that respond rapidly to the appearance of target mRNA and are then degraded or turned off when the target mRNA is degraded. Conclusion In this work, we have reported quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)+-RNA locali-
Fig.6. Single-molecule images of HeLa cells transfected with poly(A)-targeting molecular beacons. For a chamber slide preparation, trypsinized HeLa cells were incubated at 37 °C with Lipofectamine 2000 and the probe for transfection and deposition on the naked glass surface for 30 min. After washing the surface with PBS, the chamber slide was assembled with a coverslip. For the single-molecule fluorescence detection using the TIR-based smFRET instrument, the chamber slide was mounted on the inverted microscope after additional incubation for the indicated time under normal culture conditions. The resulting fluorescence signals were recorded in real time with a resolution of 0.2 s per scan for 40 s. The image is split into Cy3 (<611 nm) and Cy5 (>611 nm) emission channels, each 25 50 mm. Below are time trace data of the fluorescence emission from each fluorophore, demonstrating that the single-molecule spectroscopic events of the fluorescence changes of Cy3 and Cy5 in cells clearly differ under constant laser excitation and that the fluorescence of Cy5 almost disappears within 30 min, whereas that of Cy3 is slightly intensified.
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zation in living mammalian cells, and time-resolved intracellular binding dynamics of FRET-based molecular beacons at the singlemolecule level. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection of poly(A)-RNA and revealed a distribution of the probe itself and localization of the target RNA sequence in live cells. Moreover, FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed, for which the method is expected to reveal information hidden by ensemble averaging in bulk experiments and to allow for the detection of events too rare to perturb the ensemble-averaged signal. Acknowledgments We are very grateful to Young Dong Kim (Kyung Hee University) for his kind help. This work was supported by the Basic Science Program through the National Research Foundation of Korea (KRF) funded by the Ministry of Education, Science, and Technology (MEST) (2011-0021956 and 2012-001680). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2012.07.010. References [1] G.F. Joyce, The antiquity of RNA-based evolution, Nature 418 (2002) 214–216. [2] M.P.C. van de Corput, R.W. Dirks, R.P.M. van Gijlswijk, E. van Binnendijk, C.M. Hattinger, R.A. de Paus, J.E. Landegent, A.K. Raap, Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxide-labeled oligonucleotides and tyramide signal amplification, J. Histochem. Cytochem. 46 (1998) 1249–1259.
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