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Cy5-Conjugated Hybridization-Sensitive Fluorescent Oligonucleotides for Ratiometric Analysis of Nuclear Poly(A)+ RNA Takeshi Kubota,† Shuji Ikeda,† Hiroyuki Yanagisawa,† Mizue Yuki,† and Akimitsu Okamoto*,†,‡ † ‡
RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ABSTRACT: Subnuclear poly(A)+ RNA localization in living mammalian cells was visualized by ratiometric analysis using hybridization-sensitive fluorescent oligonucleotide probes. Probes were oligonucleotides, which contained a Cy5 fluorescent dye at the strand end and a thiazole orange double-labeled nucleotide inside strand. A ratiometric analysis using poly(A)-targeting probes revealed a distribution of the probe itself as red fluorescence and localization of the target RNA sequence in cell nuclei as green fluorescence. The fluorescence of the subnuclear poly(A)+ RNA hybridized with the poly(A)-targeting probes was observed as puncta in interchromatin areas.
’ INTRODUCTION mRNAs are transcribed from active chromosomal loci and spliced or edited at distinct domains known as speckles,1,2 and then transported to nuclear membranes by specific proteins to pass through a nuclear pore.3,4 Effective monitoring of the expression and behavior of mRNA in a cell nucleus is significant for cell biologists. To detect subnuclear mRNA localization using a fluorescence signal, development of fluorescent probes binding with the poly(A) sequence of mRNA may be one of the most effective approaches. Much information regarding transcription and translocation in cells has already been provided by poly(A)+ RNA imaging.5,6 However, conventional probes, which are simply labeled with fluorescent dyes, may 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 probes and the localization of their target RNA is important; thus, development of probes with properties allowing this simultaneous observation is required. The latest hybridization-sensitive fluorescent probes, which possess an ability for fluorescence to be emitted only when they are hybridized with the target RNA, have been reported.79 However, they often require the formation of higher-order structures or covalent bonds, which is disadvantageous in living cells. In this article, we report the design of a hybridization-sensitive fluorescent probe with a reference fluorophore for more effective detection of poly(A)+ RNA localization. Addition of a red r 2011 American Chemical Society
fluorescent dye, Cy5, as a reference signal for monitoring probe distribution to probes with a hybridization-sensitive fluorescence emission function served visualization of not only the distribution of the probe itself, but also its target RNA localization in living cells.
’ EXPERIMENTAL PROCEDURES Synthesis of a Cy5-Conjugated ECHO Probe. Probes were synthesized using a conventional phosphoramidite method. A diamino-modified nucleoside phosphoramidite (a precursor of D514) was synthesized as described in our previous paper.10 Cy5 of the 50 end was incorporated using Cy5 phosphoramidite (Glen Research), of which one hydroxy group was protected with a 4-monomethoxytrityl group. The synthesized probes were cleaved from the support and deprotected (except for the Cy5 monomethoxytrityl group) in 28% aqueous ammonia at 25 °C for 8 h. After removal of ammonia under reduced pressure, the unlabeled probes were purified by reversed-phase HPLC on a 5-ODS-H column (10 mm 150 mm, eluted with a solvent mixture of 0.1 M triethylammonium acetate (TEAA), pH 7.0, in a linear gradient of from 5% to 60% acetonitrile over 30 min at a flow rate of 3.0 mL/min). A solution of a succinimidyl ester of the thiazole orange dye (50 equiv per active amino group of the synthesized oligomer) in DMF was added to a solution of the unlabeled probe in 100 mM sodium carbonate buffer (pH 9.0) and incubated at 25 °C for 30 min.10 The reaction mixture was Received: April 12, 2011 Revised: July 11, 2011 Published: July 14, 2011 1625
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Bioconjugate Chemistry treated with ethanol (80 °C, 30 min) to precipitate the reaction products. After centrifugation at 4 °C for 30 min, the supernatant was removed. The pellet was incubated in 500 μL of 20% acetic acid at 25 °C for 20 min to deprotect the Cy5 monomethoxytrityl group. After extraction three times with ethyl acetate, the aqueous phase was passed through a 0.45 μm filter. The product was purified by reversed-phase HPLC on a 5-ODS-H column (10 mm 150 mm, elution with a solvent mixture of 0.1 M TEAA, pH 7.0, in a linear gradient of from 5% to 37.5% acetonitrile over 30 min at a flow rate of 3.0 mL/min). The synthesized probes were identified by MALDI-TOF mass spectrometry. (Here, the molecular weight of the counteranions of one Cy5 and two D514 dyes was not included in the value of M.): ProbeU, calcd. for C329H431N54O198P24S2, 9117.7 [M 2H]+, found 9118.5; ProbeA, calcd. for C350H452N117O156P24S2, 9601.5 [M 2H]+, found 9599.7. The probe concentration was determined by absorption at 260 nm with a UV-2550 spectrophotometer (Shimadzu). Fluorescence Spectra. Fluorescence spectra of the probes (0.5 μM) were measured in a HEPES-buffered solution (120 mM potassium chloride, 5 mM sodium chloride, 25 mM HEPES, pH 7.2), using a quartz cuvette with a 1 cm path length at 25 °C. The excitation and emission bandwidths were 1.5 nm. D514 and Cy5 of probes were excited at 514 and 633 nm, respectively. Spectra were recorded using a RF-5300PC spectrofluorophotometer (Shimadzu). Cell Culture. Culture reagents were purchased from Invitrogen. HeLa cells and COS7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin, and 50 mg/mL streptomycin. MadinDarby canine kidney (MDCK) cells were cultured in MEM containing 10% heat-inactivated FBS, 0.1 mM nonessential amino acids, 50 U/mL penicillin, and 50 mg/mL streptomycin. NIH3T3 cells were cultured in DMEM containing 10% heat-inactivated calf serum, 50 U/mL penicillin, and 50 mg/ mL streptomycin. All cells were cultured at 37 °C under a humidified atmosphere of 5% CO2 in air. HeLa cells were a gift from Dr. Shinichi Nakagawa (RIKEN Advanced Science Institute). Other cell lines were provided by the RIKEN BioResource Center through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), Japan. For experimental use, cells (passage numbers 59) were cultured in glass-based dishes. Prior to microscope observation, the culture medium was washed and exchanged to phenol red free DMEM. Transfection and Fluorescence Imaging. The Cy5-conjugated hybridization-sensitive fluorescent probe (50 pmol) in water were introduced using 2 μL Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. After 1 h incubation at 37 °C with the Lipofectamine 2000 and a probe, the cells were washed three times with PBS and observed in phenol red free serum-supplemented DMEM. The cells were maintained under culture conditions using an incubation system during fluorescence imaging. For DAPI staining, the probetransfected cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and subsequently, after washing three times with PBS, the cells were stained with 0.5 μg/mL DAPI in PBS for 10 min at room temperature. Fluorescence images were acquired with a motorized inverted microscope (Axio Observer Z1; Zeiss) equipped with an EM-CCD (Evolve, Roper) and a 63 (oil immersion NA 1.4) objective and filter sets (ex 500/2425, dm 520, em 542/2527 for D514, ex
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575625, dm 645, em 660710 for Cy5, ex 365, dm 396, em 445/50 for DAPI). 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.
’ RESULTS AND DISCUSSION Design and Synthesis. A fluorescent double-labeled nucleotide probe has recently been developed to achieve high fluorescence intensity for a hybrid with its target nucleic acid, and effective quenching of the unhybridized probe.10,11 The main mechanism for the effective fluorescence suppression is based on exciton coupling theory,;14 thus, the probe is known as an exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probe. Fluorescence switching depending on hybridization with their target RNA would be useful for cellular RNA detection.15,16 However, ECHO probes have the disadvantage that the detection of the distribution of the probe itself within a cell is difficult because they usually show very weak fluorescence in the absence of a target RNA, although this is also an advantage because the background fluorescence arising from an excess of probes in the cell is inconspicuous. Therefore, installation of a fluorescence indicator into the probe to show its distribution is required for RNA localization analysis with a fair degree of precision. We designed a new ECHO probe to monitor both the distribution of the probe and localization of the target RNA using ratiometric analysis. A green fluorescent nucleotide, D514, in ECHO probes was adopted for hybridization-sensitive fluorescence emission and 20 -O-methylribose nucleotides were used for ECHO probes (except D514) to increase resistance to intracellular nucleases.1719 In addition, a red fluorescent dye, Cy5, was attached as a reference signal to the 50 end of the ECHO probes. The two dyes were spatially separated in the probe using two hexaethylene glycol spacers and fifteen nucleotides. One of the Cy5-conjugated ECHO probes, ProbeU, has a sequence Cy5-s-s-U15D514U6, where ‘s’ denotes a hexaethylene glycol and ‘U’ is 20 -O-methyluridine. This probe is expected to recognize a repeated-adenine sequence, e.g., a poly(A) tail of mRNA (Figure 1). We also synthesized ProbeA, Cy5-s-s-A15D514A6 (where A is 20 -O-methyladenosine), as a control probe. Fluorescence of Probes. Cy5-conjugated ECHO probes exhibited onoff switching of fluorescence at the D514 emission wavelength depending on addition of the complementary RNA strand. The emission spectrum of single-stranded ProbeU (500 nM) excited at 514 nm showed a negligible fluorescence in HEPES buffer, whereas ProbeU hybridized with poly(A) RNA displayed much stronger D514 fluorescence (29-fold increase at 532 nm) (Figure 2a). On the other hand, the fluorescence spectra of the ProbeU excited at 633 nm almost overlapped, regardless of hybridization with poly(A) RNA (within 1.01 fold at 660 nm). The fluorescence spectra of ProbeA also showed similar emission properties (Figure 2b). Fluorescence intensity of D514 and Cy5 in ProbeU was also measured in various probe concentrations from 500 nM to 50 nM in 50 nM steps. The fluorescence decreased proportionally and the ratio of the fluorescence intensities of D514 and Cy5 was almost constant through the experiment (Figure 2c and d). The Cy5 fluorescence intensity of ProbeU exhibited a linear relationship with the amount of ProbeU at a constant concentration of A22 RNA even in the presence of excess probe (Figure 3). 1626
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Figure 1. Sequence and structure of Cy5-conjugated ECHO probe (probeU). ‘s’ denotes a hexaethylene glycol linker.
In contrast, the D514 fluorescence intensity of a ProbeU-containing solution increased in proportion to the amount of added ProbeU until the ProbeU/A22 RNA ratio became 1:1, but it almost reached a plateau when the concentration of ProbeU exceeded that of A22 RNA. The fluorescence behavior of the probe, where both a hybridization-sensitive fluorescent dye (D514) and a hybridization-insensitive fluorescent dye (Cy5) were installed into a single nucleic acid strand, suggested its strong potential as a probe for ratiometric fluorescence analysis simultaneously visualizing both the distribution of the probe and the location of the target poly(A)+ RNA in a cell. In addition, the fluorescence of Cy5 regardless of hybridization and the proportional change in the probe fluorescence intensities depending on probe concentration suggested that the Cy5 fluorescence of the probe would be effective as a standard for the normalization of fluorescence intensity in the inter- and intracellular D514 fluorescence comparison experiments. RNA Localization and Probe Distribution in HeLa Cells. Having established the fluorescence behavior of Cy5-conjugated ECHO probes in vitro, we examined the fluorescence imaging of human cancer cells, HeLa cells, where a Cy5-conjugated ECHO probe was transfected. ProbeU (50 pmol) was transfected into HeLa cells by 1 h incubation with Lipofectamine 2000, and then the fluorescence emission from cells was examined using a green filter set (ex 500/2425, dm 520, em 542/2527) and a red filter set (ex 575625, dm 645, em 660710). The fluorescence images from D514 and Cy5 of ProbeU in the cells appeared different. Cy5 fluorescence, which indicates distribution of the probe, was strong and uniform in nuclei except for nucleoli, whereas relatively weaker fluorescence was observed
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Figure 2. Fluorescence of Cy5-conjugated ECHO probes. (a) Fluorescent spectra of ProbeU (500 nM). The spectra of the single-stranded probe (ss) and hybrid with 2 equiv of poly(A) RNA (ds) were measured in 5 mM sodium chloride, 120 mM potassium chloride, and 25 mM HEPES (pH 7.2)KOH at 25 °C. Green, fluorescence spectra of ProbeU D514 excited at 514 nm; red, spectra of ProbeU Cy5 excited at 633 nm. (b) Fluorescent spectra of ProbeA (500 nM). (c) Change in the fluorescence intensities of D514 and Cy5 in the hybrid of ProbeU and poly(A) RNA depending on ProbeU concentration. The concentration of ProbeU was changed from 500 nM to 50 nM in 50 nM steps. (d) Relationship between ProbeU D514 and Cy5 fluorescence intensities. The fluorescence intensities of D514 at 530 nm and Cy5 at 660 nm in the spectra in (c) were plotted. There was a linear relationship among different probe concentrations (R2 = 0.995).
Figure 3. Change in the fluorescence intensities of D514 and Cy5 in the hybrid of ProbeU and A22 RNA depending on ProbeU concentration. The fluorescence intensity of ProbeU in the presence of A22 RNA (0.2 μM) was measured in 50 mM sodium phosphate (pH = 7.0) and 100 mM sodium chloride. The concentration of ProbeU was changed from 0 μM to 0.5 μM in 0.1 μM steps. Green circles, fluorescence intensities of ProbeU D514 at 532 nm, excited at 514 nm; red triangles, fluorescence intensities of ProbeU Cy5 at 660 nm, excited at 633 nm.
in the cytoplasmic area (Figure 4a). The fluorescence from the nucleoli, whose shape can be confirmed in DIC images, showed weak fluorescence, suggesting that almost all of the probe was excluded from nucleoli. The nucleolus is a subnuclear organelle in which rRNA is transcribed and assembled, and thus, it may be difficult for the probe to penetrate the highly rRNA-accumulated 1627
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Figure 4. Fluorescence images of probeU-transfected HeLa cells. (a) Cell images. The fluorescence from D514 and Cy5 of ProbeU was acquired with green and red filter sets, respectively. Bar: 10 μm. (b) Puncta emerging in the nucleus. Fluorescent intensities along the white line drawn in the DIC image (right) were plotted. Green, ProbeU D514; red, Cy5. Bar: 10 μm.
nucleolus area. On the other hand, D514 fluorescence, which indicates localization of poly(A)+ RNA through probe hybridization, was mainly observed in nuclei as clear puncta. A line profile showing the fluorescence intensities of D514 and Cy5 of ProbeU in a HeLa cell showed the striking difference of fluorescence distribution seen in Figure 4b. The fluorescence arising from D514 exhibited three sharp signal peaks (arrowheads 1, 2, and 3) along the line indicated in the DIC image, whereas the fluorescence intensity of Cy5 was stronger, but gave a profile with blunted responses. The fluorescence profile of ProbeU Cy5 in a HeLa cell suggests that the cell nucleus was filled with the probe and the probe D514 fluorescence showed the localization of the poly(A)+ RNA hybridized with the probe. The fluorescence image of the D514 of ProbeU was also quite different from the image of the chromatin structure stained with DAPI. When the chromatin structures of HeLa cells were stained with DAPI (0.5 μg/mL) after the probe transfection process into
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Figure 5. DAPI staining of ProbeU-transfected HeLa cells. (a) Cell images. ProbeU-transfected cells were stained with DAPI after being fixed with 4% paraformaldehyde. Green, fluorescence from D514; blue, fluorescence from DAPI. Bar: 10 μm. (b) Distinct patterns of the ProbeU D514 and DAPI fluorescence images. Fluorescence intensities along the white line drawn in the DIC image of (a) were plotted. Green, ProbeU D514; blue, DAPI. Filled arrowheads, ProbeU D514-predominant region; open arrowheads, DAPI-predominant region.
the cells was fixed with paraformaldehyde, the fluorescence signals arising from the D514 of ProbeU and DAPI appeared alternately in the cell nucleus (Figure 5a). The region stained by DAPI is a DNA condensed region, and it is also a region with a relatively low transcription activity.20,21 On the other hand, the region stained by ProbeU D514 probably has a relatively high transcription activity because D514 emits strong fluorescence when the probe is hybridized with poly(A)+ RNA. The line profile of the fluorescence intensities in the cell clearly indicates the different distributions of DNA and poly(A)+ RNA represented by mRNA (Figure 5b). Open arrowheads in the figure show the regions where the signal from DNA (i.e., fluorescence of DAPI) is predominant and filled arrowheads indicate the regions where the signal from poly(A)+ RNA (i.e., fluorescence of hybridized ProbeU) is predominant. The distribution of fluorescence suggests that poly(A)+ RNA was detected in interchromatin spaces, as reported by Politz et al.5 The fluorescence image obtained from the ProbeA-transfected HeLa cells also explained the fluorescence property of Cy5conjugated ECHO probes. ProbeA, which can be hybridized with RNA containing a U6AU15 sequence, seems to have only a small 1628
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Poly(A)+ RNA Localization in Other Cell Types. The ratio-
metric RNA analysis using Cy5-conjugated ECHO probes also facilitated the observation of a similar poly(A)+ RNA localization pattern in other mammalian cell types. We tested the fluorescence imaging for NIH3T3 (mouse), MDCK (dog), and COS7 (monkey) cells, which were transfected according to the ProbeU lipofection protocol (efficiency of lipofection was 3060%). The fluorescence of ProbeU Cy5 was observed to be strongly uniform in the nuclei of these cells except for in their nucleoli, whereas several puncta appeared in the fluorescence of the ProbeU D514 (Figure 7). Figure 6. Fluorescence images of ProbeU- or ProbeA-transfected HeLa cells.
’ CONCLUSION An oligonucleotide, which contained a Cy5 fluorescent dye at the strand end and a thiazole orange double-labeled nucleotide at the strand inside was designed for fluorescence imaging of poly(A)+ RNA in a cell. Subnuclear poly(A)+ RNA localization in living mammalian cells was visualized by a ratiometric analysis using the Cy5-conjugated ECHO probe. Ratiometric analysis using the poly(A)-targeting probe revealed the distribution of the probe itself as a red fluorescence and localization of target RNA sequence in cell nuclei as a green fluorescence. The fluorescence of the subnuclear poly(A)+ RNA hybridized with the poly(A)targeting probe was observed as puncta in interchromatin areas. ’ AUTHOR INFORMATION Corresponding Author
*Phone: (+81) 48-467-9238. Fax: (+81) 48-467-9205. E-mail:
[email protected].
Figure 7. Fluorescence images of ProbeU in various mammalian cell types. ProbeU was transfected into NIH3T3, MDCK, and COS7 cells according to the protocol for transfection into HeLa cells. Bar: 10 μm.
chance of binding with RNA in cells. When ProbeA was transfected into HeLa cells, cell uptake of the probe was confirmed by the fluorescence of Cy5. The fluorescence of ProbeA Cy5 distributed mainly in the nuclei, which was strong and uniform (Figure 6). Cy5 fluorescence indicates the distribution of all ProbeA in the cell, which contains excess probes, and it is regardless of hybridization with RNA. The fluorescence distribution pattern observed for ProbeA Cy5 was similar to that of ProbeU Cy5. In contrast, the distribution of the fluorescence of ProbeA D514 was different from that of ProbeU D514. D514 fluorescence arises from hybridization with the complementary RNA; i.e., the distribution of the fluorescence of D514 in cell indicates the localization of the complementary RNA. The fluorescence arising from D514 of ProbeA was much weaker than that from ProbeU, and no punctum was observed in the nucleus. Although the reason for the weak nonspecific fluorescence emission of ProbeA remains unknown, the fluorescence was suppressed in the cell, compared with that of ProbeU. In contrast, the puncta with D514 fluorescence observed for ProbeU indicates that they are not a result of accumulation of the fluorescent probe, but an accumulation of the poly(A)+ RNA hybridized with ProbeU. The fluorescence images of Cy5-conjugated ECHO probes in the cells showed both the distribution of the probe and the RNA localization in nuclei.
’ ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Young Scientists (B) (#21,750,182) (TK) and a Grant-in-Aid for Scientific Research (B) (#20,350,083) (AO) from the Japan Society for the Promotion of Science, and a Grant-in-Aid for Scientific Research on Priority Areas (Cancer) (#20,014,030) (AO) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). ’ REFERENCES (1) Misteli, T., Caceres, J. F., and Spector, D. L. (1997) The dynamics of a pre-mRNA splicing factor in living cells. Nature 387, 523–527. (2) Lamond, A. I., and Spector, D. L. (2003) Nuclear speckles: a model for nuclear organelles. Nat. Rev. Mol. Cell Biol. 4, 605–612. (3) Tokunaga, K., Shibuya, T., Ishihama, Y., Tadakuma, H., Ide, M., Yoshida, M., Funatsu, T., Ohshima, Y., and Tani, T. (2006) Nucleocytoplasmic transport of fluorescent mRNA in living mammalian cells: nuclear mRNA export is coupled to ongoing gene transcription. Genes Cells 11, 305–317. (4) Enninga, J., Levy, D. E., Blobel, G., and Fontoura, B. M. (2002) Role of nucleoporin induction in releasing an mRNA nuclear export block. Science 295, 1523–1525. (5) Politz, J. C., Tuft, R. A., Pederson, T., and Singer, R. H. (1999) Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells. Curr. Biol. 9, 285–291. (6) Taneja, K. L., Lifshitz, L. M., Fay, F. S., and Singer, R. H. (1992) Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy. J. Cell Biol. 119, 1245–1260. 1629
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