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5-Ethynylcytidine as a new agent for detecting RNA synthesis in live cells by “click” chemistry
Analytical Biochemistry 434 (2013) 128–135
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5-Ethynylcytidine as a new agent for detecting RNA synthesis in live cells by ‘‘click’’ chemistry Dezhong Qu a,b,1, Li Zhou a,b,1, Wei Wang a,c, Zhe Wang a, Guoxin Wang a, Weilin Chi a, Biliang Zhang a,d,⇑ a
The State Key Laboratory of Respiratory Diseases, RNA Chemical Laboratory, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China b Graduate University of the Chinese Academy of Sciences, Beijing 100049, China c Guangzhou RiboBio Co., Ltd., Guangzhou Science Park, Guangzhou 510663, China d School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
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Article history: Received 29 September 2012 Received in revised form 22 November 2012 Accepted 27 November 2012 Available online 3 December 2012 Keywords: "Click" chemistry EU 5-Ethynylcytidine RNA labeling
a b s t r a c t Detection of RNA synthesis in cells to measure the rate of total transcription is an important experimental technique. To screen the best nucleoside analogue for labeling RNA synthesis, a series of alkyne-modified nucleoside analogues, including 5-ethynylcytidine (EC) and 8-ethynyladenosine (EA), were successfully synthesized by the Sonogashira coupling reaction. The synthesis of RNA or DNA was assayed based on the biosynthetic incorporation of these analogues into newly transcribed RNA or replicating DNA. Analogue-labeled cellular RNA or DNA was detected quickly and with high sensitivity via ‘‘click’’ chemistry with fluorescent azides, followed by fluorescence microscopic imaging. The results showed that EC was efficiently incorporated into RNA, but not into DNA, in seven cell lines, as also previously shown for 5-ethynyluridine (EU). Moreover, EC was able to assay transcription rates of various tissues in animals and the rate of metabolism of EC was much faster than that of EU. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.
Detection and quantification of RNA synthesis in cells is a widely used technique to monitor cell viability, health, and metabolism rate. Until recently, two approaches for direct monitoring of RNA synthesis have been used. The first method relies on incorporation of the radioisotopelabeled nucleosides, e.g., [3H]uridine, followed by tissue autoradiography [1]. Such approach is cumbersome and slow and has low sensitivity, requiring exposure times of weeks to months under conditions of complete exclusion of external light. The need to use radioactive materials also requires a higher level of experimental caution and a special laboratory setup. Therefore, many clinical and research laboratories prefer to avoid this technique [1]. The second method was developed as a nonradioactive alternative and engages incorporation into nascent RNA of uridine chemical analogues, such as 5-bromouridine (BrU)2, which can then be immunodetected using specific antibodies. BrU can be introduced ⇑ Corresponding author. Fax: +86 20 32290137. E-mail address: [email protected] (B. Zhang). These authors contributed equally to this work. 2 Abbreviations used: EC, 5-ethynylcytidine; EA, 8-ethynyladenosine; BrU, 5-bromouridine; EU, 5-ethynyluridine; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; DAPI, 40 ,6-diamidino-2-phenylindole; NMR, nuclear magnetic resonance; Cy3-azide, 2-[3-(1,3-dihydro-1,1-dimethyl-3-(6-azidohexyl)-2Hbenz[e]indol-2-ylidene)propenyl]-3,3-dimethyl-1-ethyl-3H-indolium bromide; DCM, dichloromethane; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; ip, intraperitoneally. 1
to the cells in the form of a nucleoside or as a 50 -triphosphate nucleotide. Since the cellular membrane is impermeable to 5-bromouridine triphosphate, various techniques have been used to deliver this analogue to the inside of cells, such as microinjection [2,3], membrane permeabilization [4], liposome-mediated transfection [5], scratch labeling [6], or osmotic shock [7]. In contrast, BrU can be taken up by cells spontaneously, where it is converted into 50 mono-, 50 -di-, and 50 -triphosphate derivatives by cellular kinases and subsequently incorporated into the newly synthesized RNA transcripts [8]. Although the use of BrU is safer and more convenient than that of [3H]uridine, it poses significant limitation, since the BrU antibody is large and, hence, does not penetrate the tissues. Therefore, this approach has very limited use in whole animals or intact tissues. Most recently, a new method for RNA synthesis monitoring has been developed, which involves the incorporation of 5-ethynyluridine (EU), a uridine analogue, into cellular RNA and subsequent reaction of EU with a fluorescent azide via ‘‘click’’ chemistry [9]. This approach offers big promise in the detection and quantification of RNA synthesis, since the reaction is highly reliable, efficient, and selective. Importantly, azides and alkynes are bio-orthogonal molecules and are compatible with a wide range of solvents, including water. Furthermore, fluorescent azides are very small and are just 1/500 the size of an antibody. Thus fluorescent azides demonstrate very high diffusion rate and ability to penetrate intact animal tissues effectively [9–11]. All these advantages grant the
0003-2697/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.11.023
5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135
EU-based RNA synthesis monitoring assays higher sensitivity and shorter procedural timing [12,13]. To further improve the efficiency, speed, and sensitivity of RNA synthesis detection and quantification, we have synthesized and tested a series of alkyne-modified nucleoside analogues. Upon examination of new analogue performance both in vitro and in vivo, we propose a novel, more advanced approach for monitoring of RNA synthesis in cell culture and animals.
Materials and methods HeLa (human epithelial cervical cancer cell line), C2C12 (mouse muscle myoblast cell line), HLF (human embryonic lung fibroblast cell line), and LLC (mouse Lewis lung cancer cell line) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM). A549 (human lung cancer cell line) and H1299 (human non-small-cell lung carcinoma cell line) cells were maintained in RPMI 1640 medium. HUVECs (human umbilical vein endothelial cells) were maintained in EGM-2 medium (Clonetics). All media were supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Cells were grown in an atmosphere of 5% CO2 at 37 °C. All cell lines were kindly provided by Duanqing Pei’s laboratory at the Guangzhou Institute of Biomedicine and Health (Guangzhou, China). All of the basic chemicals, such as hydroxyurea, thymidine, actinomycin D, CuSO4, ascorbic acid, paraformaldehyde, glycine, and 40 ,6-diamidino-2-phenylindole (DAPI), as well as the synthetic compounds, such as 2-(2-anilinovinyl)-1-ethyl-3,3-dimethyl-3Hindolium iodide, 5-iodouridine, 5-iodocytidine, 8-bromoadenosine, 1,1,2-trimethyl-3-(6-azidohexyl)-1H-benzo[e]indolium bromide, and trimethylsilylacetylene, were purchased from Sigma–Aldrich (St. Louis, MO, USA). Chemical synthesis General procedure 1: addition of alkynes using the Sonogashira coupling reaction Copper(I) iodide (4 mM), anhydrous triethylamine (20 ml), trimethylsilylacetylene (20 mM), and tetrakis(triphenylphosphine)palladium(0) (2 mM) were added to a degassed solution of the desired nucleoside (10 mM) in anhydrous dimethyl formamide (100 ml), and the mixture was left to stir at temperature from 25 to 50 °C. After 6 h the solvent was removed in vacuum and the residue dissolved in methanol (100 ml) and reacted with potassium carbonate (15 mM) to remove the trimethylsilyl group, yielding ethynyl-modified nucleoside analogues. Ethynyl-modified nucleoside analogues were then purified by flash chromatography on silica. 5-Ethynyluridine EU was synthesized from 5-iodouridine and trimethylsilylacetylene using the Sonogashira coupling reaction according to general procedure 1 (above). Purification by wet flash column chromatography eluting with methanol in dichloromethane (15–25%) yielded EU as a white powder (1.40 g, 52%). EU analysis: white crystals, molecular weight 268.22; ES-API LC/MS M = 268.07 (m/z), [M+H]+ = 269.1, [M+Na]+ = 291.1, [2M+Na]+ = 559.1; 1H NMR (DMSO-d6, 400 MHz): d 11.623 (s, 1H, H-NH), 8.372 (s, 1H, H-6), 5.737 (d, 1H, J = 4.8 Hz, H-10 ), 5.413 (d, 1H, J = 5.2 Hz, HO-20 ), 5.229 (t, 1H, J = 4.8 Hz, HO-30 ), 5.060 (d, 1H, J = 5.2 Hz, HO-50 ), 4.090 (s, 1H, H-8), 4.040 (m, 1H, H-20 ), 3.981 (m, 1H, H-30 ), 3.861 (m, 1H, H-40 ), 3.693–3.554 (m, 2H, H-50 ); 13C NMR (DMSO-d6, 500 MHz): d 161.612 (C-4), 149.665 (C-2), 144.658 (C-6), 97.639 (C-5), 88.417 (C-10 ), 84.758 (C-40 ), 83.609 (C-7), 76.314 (C-8), 73.973 (C-20 ), 69.330 (C-30 ), 60.222 (C-50 ).
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5-Ethynylcytidine (EC) EC was synthesized from 5-iodocytidine and trimethylsilylacetylene using the Sonogashira coupling reaction according to general procedure 1 (above). Purification by wet flash column chromatography (silica preequilibrated with 1% Et3N) eluting with methanol in dichloromethane (15–30%) yielded EC as a light yellow powder (1.28 g, 48%). EC analysis: light yellow powder, molecular weight 267.24; ES-API LC/MS M = 267.09 (m/z), [M+H]+ = 268.1, 1 [M+Na]+ = 290.1, [2M+Na]+ = 557.2; H NMR (DMSO-d6, 400 MHz): d 8.363 (s, 1H, H-6), 7.693, 6.818 (ss, 2H, H-NH2), 5.741 (d, 1H, J = 3.2 Hz, H-10 ), 5.369 (b, 1H, HO-20 ), 5.188 (t, 1H, J = 4.8 Hz, HO-30 ), 4.987 (b, 1H, HO-50 ), 4.321 (s, 1H, H-8), 3.942 (b, 2H, H-20 ,30 ), 3.842 (m, 1H, H-40 ), 3.717–3.553 (dm, 2H, H-50 ); 13 C NMR (DMSO-d6, 500 MHz): d 164.150 (C-4), 153.597 (C-2), 145.820 (C-6), 89.465 (C-10 ), 88.848 (C-5), 85.795 (C-40 ), 84.042 (C-7), 75.819 (C-8), 74.352 (C-20 ), 68.814 (C-30 ), 59.932 (C-50 ). 8-Ethynyladenosine (EA) EA was synthesized from 8-bromoadenosine and trimethylsilylacetylene using the Sonogashira coupling reaction according to general procedure 1 (above). Purification by wet flash column chromatography (silica preequilibrated with 1% Et3N) eluting with methanol in dichloromethane (20–35%) yielded EA as a light yellow powder (1.48 g, 51%). EA analysis: light yellow powder, molecular weight 291.26; ES-API LC/MS M = 291.10 (m/z), [M+H]+ = 292.1, [M+Na]+ = 314.1, [2 M+Na]+ = 605.2; 1H NMR (DMSO-d6, 400 MHz): d 8.166 (s, 1H, H-2), 7.674 (b, 2H, H-NH2), 5.946 (d, 1H, J = 6.8, H-10 ), 5.538 (m, 1H, HO-20 ), 5.455 (d, 1H, J = 6.4 Hz, HO-30 ), 5.224 (d, 1H, J = 4.4 Hz, HO-50 ), 5.024 (m, 1H, H-20 ), 5.003 (s, 1H, H-11), 4.184 (m, 1H, H-30 ), 3.986 (m, 1H, H40 ), 3.664–3.527 (dm, 2H, H-50 ); 13C NMR (DMSO-d6, 500 MHz): d 156.263 (C-6), 153.572 (C-2), 148.415 (C-4), 133.026 (C-8), 119.026 (C-5), 89.438 (C-10 ), 87.372 (C-40 ), 86.383 (C-10), 72.743 (C-11), 71.584 (C-20 ), 71.121 (C-30 ), 62.261 (C-50 ). Synthesis of 2-[3-(1,3-dihydro-1,1-dimethyl-3-(6-azidohexyl)-2Hbenz[e]indol-2-ylidene)propenyl]-3,3-dimethyl-1-ethyl-3H-indolium bromide (Cy3-azide) 2-(2-Anilinovinyl)-1-ethyl-3,3-dimethyl-3H-indolium iodide (5 g, 12 mmol) was dissolved in acetic anhydride (25 ml) and pyridine (25 ml) and stirred for 30 min. 1,1,2-Trimethyl-3-(6-azidohexyl)-1H-benzo[e]indolium bromide (5.4 g, 13 mmol) was added and the mixture was heated at 110 °C for 10 h. After the solvent was evaporated, the residue was dissolved in 100 ml of dichloromethane (DCM), washed with water, and dried over sodium sulfate. After the solvent was evaporated, the residue was purified by silica-gel column chromatography with DCM/methanol (15:1) to yield the product as a purple solid (2.0 g, yield 28.0%). 1H NMR (CDCl3, 400 Hz): d 1.46–1.70 (11H, m), 1.74 (6H, s), 2.00 (6H, s), 3.28 (2H, t, J = 6.80 Hz), 4.29–4.42 (4H, m), 7.11 (1.0H, d, J = 7.80 Hz), 7.20–7.63 (9H, m), 7.95 (2H, d, J = 8.60 Hz), 8.09 (1H, d, J = 8.44 Hz); 13C NMR (CDCl3, 500 Hz): d 12.97, 26.37, 26.55, 27.77, 27.85, 28.06, 28.60, 40.11, 45.01, 48.72, 50.59, 51.43, 104.31, 104.42, 110.60, 110.74, 121.80, 122.07, 124.99, 125.09, 127.78, 128.03, 128.81, 130.16, 130.75, 131.88, 133.46, 139.48, 140.71, 141.86, 150.10, 172.74, 175.20. HRMS (ESI) calcd for C35H42N5 [M]+: 532.3440. Found: 532.3444. RNA labeling using alkyne-modified nucleoside analogues in cultured cells HeLa cells were grown on glass coverslips in DMEM supplemented with 10% FBS. EU and EC were added to the complete culture medium from a 100 mM stock in H2O. EA was added to the complete culture medium from a 200 mM stock in DMSO.
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After exposure to nucleoside analogues for 4 h, all cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, then incubated with glycine at the concentration of 2 mg/ml in PBS to stop the fixing action of paraformaldehyde, and finally permeabilized by 0.2% Triton X-100 in PBS. In the case of EC, the analogue was added to the complete culture medium from a 100 mM stock in H2O and rinsed once with PBS, followed by click staining using Cy3-azide. To evaluate the labeling effect on DNA synthesis when it was inhibited, cells were incubated for 4 h with 1 mM EU, 1 mM EC, and 5 mM EA, respectively, in the presence or absence of 10 mM hydroxyurea or 2 mM thymidine. To examine the labeling effect on RNA synthesis when it was inhibited, HeLa cells were grown for 4 h in complete medium with 1 mM EU, 1 mM EC, or 5 mM EA, with or without actinomycin D (100 nM or 2 lM). The cells were then rinsed, fixed, and processed for azide coupling and DAPI staining. To evaluate the synchronous inhibition of DNA and RNA synthesis, cells were incubated for 4 h with 5 mM EA in the presence or absence of 10 mM hydroxyurea and 2 lM actinomycin D or 2 mM thymidine and 2 lM actinomycin D.
Alkyne-modified nucleoside analogue detection by click chemistry All the steps of alkyne-modified nucleoside analogue detection were performed at room temperature. The fixed cells were rinsed with PBS and stained for 30 min at room temperature with 100 mM Tris (pH 8.5), 1 mM CuSO4, 10 lM fluorescent Cy3-azide (from a 10 mM stock solution in DMSO), and 100 mM ascorbic acid (added last from a 0.5 M stock in water). After treatment with alkyne-modified nucleoside analogues, cells were washed two times with methanol, once with PBS, and then stained with DAPI. The resulting labeled and stained cells were imaged by fluorescence microscopy. EC treatment of C2C12, HLF, LLC, A549, H1299, and HUVEC cells C2C12, HLF, and LLC cells were grown on glass coverslips in DMEM supplemented with 10% FBS. A549 and H1299 cells were grown on glass coverslips in 1640 supplemented with 10% FBS. HUVECs were grown on glass coverslips in EGM-2 supplemented with 5% FBS. The cells were then incubated with 1 mM EC for
Fig.1. Label on nascent RNA in cells using alkyne-modified nucleoside analogues. (A) Schematic diagram of the click reaction for detecting alkyne-modified nucleoside analogues incorporated into cellular RNA. The terminal alkyne groups of bases readily react with an organic fluorescent azide via a Cu(I)-catalyzed [3 + 2] cycloaddition reaction (‘‘click’’ chemistry). (B1–B4) Structures of the fluorescent azide and the alkyne-modified nucleoside analogues used in this study.
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4 h, rinsed, fixed, and stained with Cy3-azide and DAPI. The resulting cells were imaged by fluorescence microscopy. EC and EU labeling of mouse tissues The 3-week-old mice were injected intraperitoneally (ip) with 2 mg EC or EU in 0.1 ml PBS. A control littermate mouse was injected ip with 0.1 ml PBS. The mouse liver, kidney, spleen, colon, and ileum were harvested 5 or 24 h after injection. Pieces of the harvested tissues were embedded in paraffin and sectioned. After paraffin removal and rehydration, the sections were stained with Cy3-azide and DAPI using the same protocol as for cultured cells (above). Upon staining, the samples were mounted for fluorescence microscopy. Results and discussion Alkyne-modified nucleoside analogues as transcriptional labels To develop a new approach for monitoring RNA synthesis via "click" reaction in cells (Fig. 1A), we synthesized a series of
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alkyne-modified nucleoside analogues including EU, EC, and EA (Fig. 1B2–B4) using the Sonogashira coupling reaction as previously described [14–19]. To be used as a detecting agent for these analogues, we synthesized a fluorescent azide derivative, Cy3azide (Fig. 1B1). HeLa cells were cultured with 1 mM EU, 1 mM EC, and 5 mM EA for 4 h, respectively, and then fixed and subsequently stained with Cy3-azide and DAPI. Last, the cells were imaged by fluorescence microscopy at excitation wavelengths of 488 and 543 nm for DAPI and Cy3, respectively. The first row of each figure shows the newly nascent RNA transcripts or newly replicated DNA labeled by nucleoside analogues with Cy3-azide (red); the second row of each figure shows the nucleus labeled by DAPI (blue); the third row of each figure shows the overlay of red and blue. Untreated HeLa cells were used as a control. The labeling pattern with EC was similar to that obtained with EU (Supplementary Fig. S1, ii and iii). All cells were uniformly labeled, with EC-labeled cells also producing strong signals for nucleoli, the cellular compartment responsible for transcription of most abundant ribosomal RNA. Upon incubation with EA, the cells showed strong staining in both nuclei and cytoplasm (Supplementary Fig. S1, iv).
Fig.2. Monitoring EC incorporation into nascent RNA in cultured HeLa cells using fluorescence microscopy. HeLa cells were incubated with 10 mM hydroxyurea, 2 mM thymidine, and 100 nM or 2 lM actinomycin D in the presence of 1 mM EC or EU for 4 h. The cells were fixed, treated with Cy3-azide and DAPI, washed, and imaged by fluorescence microscopy. (A) EC. (B) EU.
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EC labels cellular RNA, but not DNA To exert the RNA labeling effect, EC needs to enter the ribonucleoside salvage pathway and be metabolized into its triphosphate form in three subsequent phosphorylation steps: EC ? ECMP ? ECDP ? ECTP. Ribonucleotide reductase is the enzyme that catalyzes the formation of deoxyribonucleotide diphosphates from ribonucleotide diphosphates. If ECDP were a substrate for ribonucleotide reductase, the intermediate EdCDP would be phosphorylated to its triphosphate form (EdCTP), which, in its turn, would be used in the synthesis of DNA as well. Such outcome would limit the utility of the EC-based labeling procedure, since both the nas-
cent replicating DNA and the transcribed RNA would be labeled, thus making it difficult to distinguish and differentiate the two processes. To exclude such possibility, we next tested whether addition of EC to cells in culture would result in DNA labeling. Hydroxyurea and thymidine are the two well-characterized inhibitors of ribonucleotide reductase. Upon exposure to cells, they cause decreased production of the deoxyribonucleotide diphosphates, thus interfering with synthesis of deoxyribonucleotides and hampering cell cycle progression into the S phase. On the other hand, actinomycin D is widely used as a tool to inhibit, evaluate, and study transcription. Upon cell incubation (for 4 h) with EC in the presence of hydroxyurea (Fig. 2A, iii) or thymidine (Fig. 2A,
Fig.3. EC and EU detection in various tissues of animals. Mice were injected ip with 2 mg of EC or EU. Organs were harvested 5 or 24 h after injection. The organ sections were treated with Cy3-azide and DAPI, washed, and then imaged by fluorescence microscopy. (A1-A2) Distinct EC- and EU-generated signals in liver, colon, and ileum, but not kidney sections, detected 5 h after injection. (B1-B2) 24 h after injection, the EC-generated signal decreased sharply, while the EU-generated weakened signal still could be detected in liver, colon, and ileum. (C) The control results of various tissues harvested 5 h after injection.
5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135
iv), the intensity and subcellular pattern of EC stain were indistinguishable from those obtained after incubation with EC alone (Fig. 2A, ii). The results obtained with EC were also similar to those obtained with EU (Fig. 2B, ii–iv), demonstrating that EC did not incorporate into replicating DNA at substantial levels. To further demonstrate that EC could label cellular RNA, but not DNA, cells were treated with EC in the presence of actinomycin D at various concentrations. It was well documented that the presence of actinomycin D at low concentrations inhibited RNA polymerase I activity, predominantly responsible for transcription and processing of highly abundant rRNA in the nucleoli compartment of the nucleus [20]. Consistent with EC’s ability to incorporate and label nascent rRNA transcripts, the intense staining of nucleoli was abolished at the low concentration of actinomycin D (while leaving the EC signal in the rest of the cell unchanged; Fig. 2A, v), thus confirming that the original EC staining of nucleoli
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indeed represented the RNA polymerase I transcripts, most probably the pre-rRNA molecules. High concentrations of actinomycin D are known to also inhibit RNA polymerase II activity. Consistent with this, the EC staining was prevented throughout the whole cell (the effect similar to that observed with EU) upon exposure to actinomycin D at high concentrations (Fig. 2A, vi, and B, vi). Taken together, these results demonstrated that EC could incorporate into RNA transcripts produced by RNA polymerases I and II, but not into the nascent DNA. Labeling of nascent nucleic acids in cells using alkynyl-substituted purine nucleosides We next tested the labeling pattern in cells treated with alkynyl-substituted purine nucleoside analogues (EA), which turned out to be very different from that of alkynyl-substituted
Fig. 3. (continued)
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Fig. 3. (continued)
pyrimidine ribonucleoside analogue (EU and EC)-treated cells, producing pronounced staining not only of nuclei, but also of the cytoplasm (Supplementary Fig. S2, ii). Inhibition of DNA synthesis by hydroxyurea or thymidine, as well as inhibition of RNA synthesis by 2 lM actinomycin D, did not change this labeling pattern (Supplementary Fig. S2, iii–vi). Even when both DNA and RNA synthases were inhibited simultaneously by incubating cells with hydroxyurea and actinomycin D or with thymidine and actinomycin D combinations, the cellular staining pattern remained the same (Supplementary Fig. S2, vii and viii). We cannot explain this result at this point and expect that additional study of the alkynylsubstituted purine nucleosides metabolism would be required to provide an explanation. In any case, these results argued that alkynyl-substituted purine nucleoside analogues could not be used for specific detection of RNA or DNA synthesis in the cells. The use of EC in various cell types Whether EC could be used to monitor synthesis of nucleic acids in other cell types, different from HeLa cells (described above), was investigated. For this, six cell types (A549, C2C12, H1299, HLF, LLC, and HUVECs) were incubated with 1 mM EC for 4 h, then fixed by 4% paraformaldehyde in PBS, and subsequently stained with fluorescent Cy3-azide and DAPI. Results demonstrated that EC was readily incorporated into all tested cell types and could be used universally to monitor RNA synthesis (Supplementary Fig. S3).
tions were subsequently stained with fluorescent Cy3-azide and DAPI, washed, and then imaged by fluorescence microscopy. The labeling results of four different organs (liver, kidney, colon, and ileum) are summarized in Fig. 3. After the 5-h time point, no obvious EC or EU staining signal could be detected in kidney (Figs. 3A1, ii and 3A2, ii) or spleen (data not shown), although a former report demonstrated that kidney tubules and a large subset of the lymphocytes in spleen showed intensive EU staining [9]. Both EC and EU produced similar labeling patterns in liver, with very intense signal coming from hepatocytes (Fig. 3A1, i, and A2, i), consistent with hepatocytes showing strong EU staining in a prior report [9]. In colon, distinct EC-generated labeling signal was detected at the tips of villi (Fig. 3A1, iii), while the overall intensity of the EC-generated signal was lower than that originating from EU (compare Fig. 3A1, iii, and A2, iii). In ileum, EC produced strong signal at the tips of villi (Fig. 3A1, iv), while the EU-generated signal came from the base of villi (Fig. 3A2, iv). No EC-generated signal could be detected in any of four evaluated organs after 24 h (Fig. 3B1). Distinct signal still could be detected in liver, colon, and ileum of EU-injected animals (Fig. 3B2, i, iii, and iv) after 24 h; however, the strength of the signal was substantially weaker than after 5 h of injection. In ileum, the EU labeling shifted from the base of the villi toward their tips (compare Fig. 3A2, iv, and B2, iv). In summary these results demonstrate that EC was well suited to detecting RNA synthesis in proliferating tissues and that the metabolic rate of EC was faster than that of EU.
Monitoring RNA synthesis in animals using EC Conclusions To determine whether EC could be used to label RNA in animals, two 3-week-old mice were injected ip with 2 mg of EC in PBS. An uninjected littermate was used as a control. A parallel experiment was conducted with EU to compare the efficacy of labeling and the labeling patterns. Animals were euthanized and tissues collected and fixed at 5- and 24-h time points after injection. Paraffin sec-
We have demonstrated that 5-ethynylcytidine (EC), a novel cytidine analogue, could be used efficiently to monitor RNA synthesis in vitro and in vivo when coupled with Cy3-azide via a Cu(I)-catalyzed click reaction. EC demonstrated sensitivity comparable to that of EU and higher metabolic rate than EU. In contrast to
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EC, alkynyl-substituted purine nucleosides failed to label specifically the newly transcribed RNA. We predict that EC will have applications for monitoring of RNA synthesis and assaying the turnover of transcripts both in vitro and in vivo. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 30870535 and 90913017), the Combination Project of Guangdong Province and the Ministry of Education (No. 2009B090300080 and 2011B090400478), the Introduced Innovative R&D Team Program of Guangdong Province (No. 201001Y0104789252), and the 863 Program of China (No. 2012AA022501). 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.2012.11.023. References [1] M. Uddin, G.G. Altmann, C.P. Leblond, Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine, J. Cell Biol. 98 (1984) 1619–1629. [2] D. Wansink, W. Schul, I. van der Kraan, B. van Steensel, R. van Driel, L. de Jong, Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus, J. Cell Biol. 122 (1993) 283– 293. [3] D. Cmarko, P.J. Verschure, T.E. Martin, M.E. Dahmus, S. Krause, X.D. Fu, R. van Driel, S. Fakan, Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection, Mol. Biol. Cell 10 (1999) 211–223. [4] X. Wei, S. Somanathan, J. Samarabandu, R. Berezney, Three-dimensional visualization of transcription sites and their association with splicing factorrich nuclear speckles, J. Cell Biol. 146 (1999) 543–558. [5] G. Haukenes, A.M. Szilvay, K.A. Brokstad, A. Kanestrøm, K.H. Kalland, Labeling of RNA transcripts of eukaryotic cells in culture with BrUTP using a liposome transfection reagent (DOTAP), Biotechniques 22 (1997) 308–312. [6] N. Sadoni, D. Zink, Nascent RNA synthesis in the context of chromatin architecture, Chromosome Res. 12 (2004) 439–451.
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5-Ethynylcytidine as a new agent for detecting RNA synthesis in live cells by ‘‘click’’ chemistry Dezhong Qu a,b,1, Li Zhou a,b,1, Wei Wang a,c, Zhe Wang a, Guoxin Wang a, Weilin Chi a, Biliang Zhang a,d,⇑ a
The State Key Laboratory of Respiratory Diseases, RNA Chemical Laboratory, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China b Graduate University of the Chinese Academy of Sciences, Beijing 100049, China c Guangzhou RiboBio Co., Ltd., Guangzhou Science Park, Guangzhou 510663, China d School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
a r t i c l e
i n f o
Article history: Received 29 September 2012 Received in revised form 22 November 2012 Accepted 27 November 2012 Available online 3 December 2012 Keywords: "Click" chemistry EU 5-Ethynylcytidine RNA labeling
a b s t r a c t Detection of RNA synthesis in cells to measure the rate of total transcription is an important experimental technique. To screen the best nucleoside analogue for labeling RNA synthesis, a series of alkyne-modified nucleoside analogues, including 5-ethynylcytidine (EC) and 8-ethynyladenosine (EA), were successfully synthesized by the Sonogashira coupling reaction. The synthesis of RNA or DNA was assayed based on the biosynthetic incorporation of these analogues into newly transcribed RNA or replicating DNA. Analogue-labeled cellular RNA or DNA was detected quickly and with high sensitivity via ‘‘click’’ chemistry with fluorescent azides, followed by fluorescence microscopic imaging. The results showed that EC was efficiently incorporated into RNA, but not into DNA, in seven cell lines, as also previously shown for 5-ethynyluridine (EU). Moreover, EC was able to assay transcription rates of various tissues in animals and the rate of metabolism of EC was much faster than that of EU. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.
Detection and quantification of RNA synthesis in cells is a widely used technique to monitor cell viability, health, and metabolism rate. Until recently, two approaches for direct monitoring of RNA synthesis have been used. The first method relies on incorporation of the radioisotopelabeled nucleosides, e.g., [3H]uridine, followed by tissue autoradiography [1]. Such approach is cumbersome and slow and has low sensitivity, requiring exposure times of weeks to months under conditions of complete exclusion of external light. The need to use radioactive materials also requires a higher level of experimental caution and a special laboratory setup. Therefore, many clinical and research laboratories prefer to avoid this technique [1]. The second method was developed as a nonradioactive alternative and engages incorporation into nascent RNA of uridine chemical analogues, such as 5-bromouridine (BrU)2, which can then be immunodetected using specific antibodies. BrU can be introduced ⇑ Corresponding author. Fax: +86 20 32290137. E-mail address: [email protected] (B. Zhang). These authors contributed equally to this work. 2 Abbreviations used: EC, 5-ethynylcytidine; EA, 8-ethynyladenosine; BrU, 5-bromouridine; EU, 5-ethynyluridine; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; DAPI, 40 ,6-diamidino-2-phenylindole; NMR, nuclear magnetic resonance; Cy3-azide, 2-[3-(1,3-dihydro-1,1-dimethyl-3-(6-azidohexyl)-2Hbenz[e]indol-2-ylidene)propenyl]-3,3-dimethyl-1-ethyl-3H-indolium bromide; DCM, dichloromethane; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; ip, intraperitoneally. 1
to the cells in the form of a nucleoside or as a 50 -triphosphate nucleotide. Since the cellular membrane is impermeable to 5-bromouridine triphosphate, various techniques have been used to deliver this analogue to the inside of cells, such as microinjection [2,3], membrane permeabilization [4], liposome-mediated transfection [5], scratch labeling [6], or osmotic shock [7]. In contrast, BrU can be taken up by cells spontaneously, where it is converted into 50 mono-, 50 -di-, and 50 -triphosphate derivatives by cellular kinases and subsequently incorporated into the newly synthesized RNA transcripts [8]. Although the use of BrU is safer and more convenient than that of [3H]uridine, it poses significant limitation, since the BrU antibody is large and, hence, does not penetrate the tissues. Therefore, this approach has very limited use in whole animals or intact tissues. Most recently, a new method for RNA synthesis monitoring has been developed, which involves the incorporation of 5-ethynyluridine (EU), a uridine analogue, into cellular RNA and subsequent reaction of EU with a fluorescent azide via ‘‘click’’ chemistry [9]. This approach offers big promise in the detection and quantification of RNA synthesis, since the reaction is highly reliable, efficient, and selective. Importantly, azides and alkynes are bio-orthogonal molecules and are compatible with a wide range of solvents, including water. Furthermore, fluorescent azides are very small and are just 1/500 the size of an antibody. Thus fluorescent azides demonstrate very high diffusion rate and ability to penetrate intact animal tissues effectively [9–11]. All these advantages grant the
0003-2697/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.11.023
5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135
EU-based RNA synthesis monitoring assays higher sensitivity and shorter procedural timing [12,13]. To further improve the efficiency, speed, and sensitivity of RNA synthesis detection and quantification, we have synthesized and tested a series of alkyne-modified nucleoside analogues. Upon examination of new analogue performance both in vitro and in vivo, we propose a novel, more advanced approach for monitoring of RNA synthesis in cell culture and animals.
Materials and methods HeLa (human epithelial cervical cancer cell line), C2C12 (mouse muscle myoblast cell line), HLF (human embryonic lung fibroblast cell line), and LLC (mouse Lewis lung cancer cell line) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM). A549 (human lung cancer cell line) and H1299 (human non-small-cell lung carcinoma cell line) cells were maintained in RPMI 1640 medium. HUVECs (human umbilical vein endothelial cells) were maintained in EGM-2 medium (Clonetics). All media were supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Cells were grown in an atmosphere of 5% CO2 at 37 °C. All cell lines were kindly provided by Duanqing Pei’s laboratory at the Guangzhou Institute of Biomedicine and Health (Guangzhou, China). All of the basic chemicals, such as hydroxyurea, thymidine, actinomycin D, CuSO4, ascorbic acid, paraformaldehyde, glycine, and 40 ,6-diamidino-2-phenylindole (DAPI), as well as the synthetic compounds, such as 2-(2-anilinovinyl)-1-ethyl-3,3-dimethyl-3Hindolium iodide, 5-iodouridine, 5-iodocytidine, 8-bromoadenosine, 1,1,2-trimethyl-3-(6-azidohexyl)-1H-benzo[e]indolium bromide, and trimethylsilylacetylene, were purchased from Sigma–Aldrich (St. Louis, MO, USA). Chemical synthesis General procedure 1: addition of alkynes using the Sonogashira coupling reaction Copper(I) iodide (4 mM), anhydrous triethylamine (20 ml), trimethylsilylacetylene (20 mM), and tetrakis(triphenylphosphine)palladium(0) (2 mM) were added to a degassed solution of the desired nucleoside (10 mM) in anhydrous dimethyl formamide (100 ml), and the mixture was left to stir at temperature from 25 to 50 °C. After 6 h the solvent was removed in vacuum and the residue dissolved in methanol (100 ml) and reacted with potassium carbonate (15 mM) to remove the trimethylsilyl group, yielding ethynyl-modified nucleoside analogues. Ethynyl-modified nucleoside analogues were then purified by flash chromatography on silica. 5-Ethynyluridine EU was synthesized from 5-iodouridine and trimethylsilylacetylene using the Sonogashira coupling reaction according to general procedure 1 (above). Purification by wet flash column chromatography eluting with methanol in dichloromethane (15–25%) yielded EU as a white powder (1.40 g, 52%). EU analysis: white crystals, molecular weight 268.22; ES-API LC/MS M = 268.07 (m/z), [M+H]+ = 269.1, [M+Na]+ = 291.1, [2M+Na]+ = 559.1; 1H NMR (DMSO-d6, 400 MHz): d 11.623 (s, 1H, H-NH), 8.372 (s, 1H, H-6), 5.737 (d, 1H, J = 4.8 Hz, H-10 ), 5.413 (d, 1H, J = 5.2 Hz, HO-20 ), 5.229 (t, 1H, J = 4.8 Hz, HO-30 ), 5.060 (d, 1H, J = 5.2 Hz, HO-50 ), 4.090 (s, 1H, H-8), 4.040 (m, 1H, H-20 ), 3.981 (m, 1H, H-30 ), 3.861 (m, 1H, H-40 ), 3.693–3.554 (m, 2H, H-50 ); 13C NMR (DMSO-d6, 500 MHz): d 161.612 (C-4), 149.665 (C-2), 144.658 (C-6), 97.639 (C-5), 88.417 (C-10 ), 84.758 (C-40 ), 83.609 (C-7), 76.314 (C-8), 73.973 (C-20 ), 69.330 (C-30 ), 60.222 (C-50 ).
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5-Ethynylcytidine (EC) EC was synthesized from 5-iodocytidine and trimethylsilylacetylene using the Sonogashira coupling reaction according to general procedure 1 (above). Purification by wet flash column chromatography (silica preequilibrated with 1% Et3N) eluting with methanol in dichloromethane (15–30%) yielded EC as a light yellow powder (1.28 g, 48%). EC analysis: light yellow powder, molecular weight 267.24; ES-API LC/MS M = 267.09 (m/z), [M+H]+ = 268.1, 1 [M+Na]+ = 290.1, [2M+Na]+ = 557.2; H NMR (DMSO-d6, 400 MHz): d 8.363 (s, 1H, H-6), 7.693, 6.818 (ss, 2H, H-NH2), 5.741 (d, 1H, J = 3.2 Hz, H-10 ), 5.369 (b, 1H, HO-20 ), 5.188 (t, 1H, J = 4.8 Hz, HO-30 ), 4.987 (b, 1H, HO-50 ), 4.321 (s, 1H, H-8), 3.942 (b, 2H, H-20 ,30 ), 3.842 (m, 1H, H-40 ), 3.717–3.553 (dm, 2H, H-50 ); 13 C NMR (DMSO-d6, 500 MHz): d 164.150 (C-4), 153.597 (C-2), 145.820 (C-6), 89.465 (C-10 ), 88.848 (C-5), 85.795 (C-40 ), 84.042 (C-7), 75.819 (C-8), 74.352 (C-20 ), 68.814 (C-30 ), 59.932 (C-50 ). 8-Ethynyladenosine (EA) EA was synthesized from 8-bromoadenosine and trimethylsilylacetylene using the Sonogashira coupling reaction according to general procedure 1 (above). Purification by wet flash column chromatography (silica preequilibrated with 1% Et3N) eluting with methanol in dichloromethane (20–35%) yielded EA as a light yellow powder (1.48 g, 51%). EA analysis: light yellow powder, molecular weight 291.26; ES-API LC/MS M = 291.10 (m/z), [M+H]+ = 292.1, [M+Na]+ = 314.1, [2 M+Na]+ = 605.2; 1H NMR (DMSO-d6, 400 MHz): d 8.166 (s, 1H, H-2), 7.674 (b, 2H, H-NH2), 5.946 (d, 1H, J = 6.8, H-10 ), 5.538 (m, 1H, HO-20 ), 5.455 (d, 1H, J = 6.4 Hz, HO-30 ), 5.224 (d, 1H, J = 4.4 Hz, HO-50 ), 5.024 (m, 1H, H-20 ), 5.003 (s, 1H, H-11), 4.184 (m, 1H, H-30 ), 3.986 (m, 1H, H40 ), 3.664–3.527 (dm, 2H, H-50 ); 13C NMR (DMSO-d6, 500 MHz): d 156.263 (C-6), 153.572 (C-2), 148.415 (C-4), 133.026 (C-8), 119.026 (C-5), 89.438 (C-10 ), 87.372 (C-40 ), 86.383 (C-10), 72.743 (C-11), 71.584 (C-20 ), 71.121 (C-30 ), 62.261 (C-50 ). Synthesis of 2-[3-(1,3-dihydro-1,1-dimethyl-3-(6-azidohexyl)-2Hbenz[e]indol-2-ylidene)propenyl]-3,3-dimethyl-1-ethyl-3H-indolium bromide (Cy3-azide) 2-(2-Anilinovinyl)-1-ethyl-3,3-dimethyl-3H-indolium iodide (5 g, 12 mmol) was dissolved in acetic anhydride (25 ml) and pyridine (25 ml) and stirred for 30 min. 1,1,2-Trimethyl-3-(6-azidohexyl)-1H-benzo[e]indolium bromide (5.4 g, 13 mmol) was added and the mixture was heated at 110 °C for 10 h. After the solvent was evaporated, the residue was dissolved in 100 ml of dichloromethane (DCM), washed with water, and dried over sodium sulfate. After the solvent was evaporated, the residue was purified by silica-gel column chromatography with DCM/methanol (15:1) to yield the product as a purple solid (2.0 g, yield 28.0%). 1H NMR (CDCl3, 400 Hz): d 1.46–1.70 (11H, m), 1.74 (6H, s), 2.00 (6H, s), 3.28 (2H, t, J = 6.80 Hz), 4.29–4.42 (4H, m), 7.11 (1.0H, d, J = 7.80 Hz), 7.20–7.63 (9H, m), 7.95 (2H, d, J = 8.60 Hz), 8.09 (1H, d, J = 8.44 Hz); 13C NMR (CDCl3, 500 Hz): d 12.97, 26.37, 26.55, 27.77, 27.85, 28.06, 28.60, 40.11, 45.01, 48.72, 50.59, 51.43, 104.31, 104.42, 110.60, 110.74, 121.80, 122.07, 124.99, 125.09, 127.78, 128.03, 128.81, 130.16, 130.75, 131.88, 133.46, 139.48, 140.71, 141.86, 150.10, 172.74, 175.20. HRMS (ESI) calcd for C35H42N5 [M]+: 532.3440. Found: 532.3444. RNA labeling using alkyne-modified nucleoside analogues in cultured cells HeLa cells were grown on glass coverslips in DMEM supplemented with 10% FBS. EU and EC were added to the complete culture medium from a 100 mM stock in H2O. EA was added to the complete culture medium from a 200 mM stock in DMSO.
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After exposure to nucleoside analogues for 4 h, all cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, then incubated with glycine at the concentration of 2 mg/ml in PBS to stop the fixing action of paraformaldehyde, and finally permeabilized by 0.2% Triton X-100 in PBS. In the case of EC, the analogue was added to the complete culture medium from a 100 mM stock in H2O and rinsed once with PBS, followed by click staining using Cy3-azide. To evaluate the labeling effect on DNA synthesis when it was inhibited, cells were incubated for 4 h with 1 mM EU, 1 mM EC, and 5 mM EA, respectively, in the presence or absence of 10 mM hydroxyurea or 2 mM thymidine. To examine the labeling effect on RNA synthesis when it was inhibited, HeLa cells were grown for 4 h in complete medium with 1 mM EU, 1 mM EC, or 5 mM EA, with or without actinomycin D (100 nM or 2 lM). The cells were then rinsed, fixed, and processed for azide coupling and DAPI staining. To evaluate the synchronous inhibition of DNA and RNA synthesis, cells were incubated for 4 h with 5 mM EA in the presence or absence of 10 mM hydroxyurea and 2 lM actinomycin D or 2 mM thymidine and 2 lM actinomycin D.
Alkyne-modified nucleoside analogue detection by click chemistry All the steps of alkyne-modified nucleoside analogue detection were performed at room temperature. The fixed cells were rinsed with PBS and stained for 30 min at room temperature with 100 mM Tris (pH 8.5), 1 mM CuSO4, 10 lM fluorescent Cy3-azide (from a 10 mM stock solution in DMSO), and 100 mM ascorbic acid (added last from a 0.5 M stock in water). After treatment with alkyne-modified nucleoside analogues, cells were washed two times with methanol, once with PBS, and then stained with DAPI. The resulting labeled and stained cells were imaged by fluorescence microscopy. EC treatment of C2C12, HLF, LLC, A549, H1299, and HUVEC cells C2C12, HLF, and LLC cells were grown on glass coverslips in DMEM supplemented with 10% FBS. A549 and H1299 cells were grown on glass coverslips in 1640 supplemented with 10% FBS. HUVECs were grown on glass coverslips in EGM-2 supplemented with 5% FBS. The cells were then incubated with 1 mM EC for
Fig.1. Label on nascent RNA in cells using alkyne-modified nucleoside analogues. (A) Schematic diagram of the click reaction for detecting alkyne-modified nucleoside analogues incorporated into cellular RNA. The terminal alkyne groups of bases readily react with an organic fluorescent azide via a Cu(I)-catalyzed [3 + 2] cycloaddition reaction (‘‘click’’ chemistry). (B1–B4) Structures of the fluorescent azide and the alkyne-modified nucleoside analogues used in this study.
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4 h, rinsed, fixed, and stained with Cy3-azide and DAPI. The resulting cells were imaged by fluorescence microscopy. EC and EU labeling of mouse tissues The 3-week-old mice were injected intraperitoneally (ip) with 2 mg EC or EU in 0.1 ml PBS. A control littermate mouse was injected ip with 0.1 ml PBS. The mouse liver, kidney, spleen, colon, and ileum were harvested 5 or 24 h after injection. Pieces of the harvested tissues were embedded in paraffin and sectioned. After paraffin removal and rehydration, the sections were stained with Cy3-azide and DAPI using the same protocol as for cultured cells (above). Upon staining, the samples were mounted for fluorescence microscopy. Results and discussion Alkyne-modified nucleoside analogues as transcriptional labels To develop a new approach for monitoring RNA synthesis via "click" reaction in cells (Fig. 1A), we synthesized a series of
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alkyne-modified nucleoside analogues including EU, EC, and EA (Fig. 1B2–B4) using the Sonogashira coupling reaction as previously described [14–19]. To be used as a detecting agent for these analogues, we synthesized a fluorescent azide derivative, Cy3azide (Fig. 1B1). HeLa cells were cultured with 1 mM EU, 1 mM EC, and 5 mM EA for 4 h, respectively, and then fixed and subsequently stained with Cy3-azide and DAPI. Last, the cells were imaged by fluorescence microscopy at excitation wavelengths of 488 and 543 nm for DAPI and Cy3, respectively. The first row of each figure shows the newly nascent RNA transcripts or newly replicated DNA labeled by nucleoside analogues with Cy3-azide (red); the second row of each figure shows the nucleus labeled by DAPI (blue); the third row of each figure shows the overlay of red and blue. Untreated HeLa cells were used as a control. The labeling pattern with EC was similar to that obtained with EU (Supplementary Fig. S1, ii and iii). All cells were uniformly labeled, with EC-labeled cells also producing strong signals for nucleoli, the cellular compartment responsible for transcription of most abundant ribosomal RNA. Upon incubation with EA, the cells showed strong staining in both nuclei and cytoplasm (Supplementary Fig. S1, iv).
Fig.2. Monitoring EC incorporation into nascent RNA in cultured HeLa cells using fluorescence microscopy. HeLa cells were incubated with 10 mM hydroxyurea, 2 mM thymidine, and 100 nM or 2 lM actinomycin D in the presence of 1 mM EC or EU for 4 h. The cells were fixed, treated with Cy3-azide and DAPI, washed, and imaged by fluorescence microscopy. (A) EC. (B) EU.
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EC labels cellular RNA, but not DNA To exert the RNA labeling effect, EC needs to enter the ribonucleoside salvage pathway and be metabolized into its triphosphate form in three subsequent phosphorylation steps: EC ? ECMP ? ECDP ? ECTP. Ribonucleotide reductase is the enzyme that catalyzes the formation of deoxyribonucleotide diphosphates from ribonucleotide diphosphates. If ECDP were a substrate for ribonucleotide reductase, the intermediate EdCDP would be phosphorylated to its triphosphate form (EdCTP), which, in its turn, would be used in the synthesis of DNA as well. Such outcome would limit the utility of the EC-based labeling procedure, since both the nas-
cent replicating DNA and the transcribed RNA would be labeled, thus making it difficult to distinguish and differentiate the two processes. To exclude such possibility, we next tested whether addition of EC to cells in culture would result in DNA labeling. Hydroxyurea and thymidine are the two well-characterized inhibitors of ribonucleotide reductase. Upon exposure to cells, they cause decreased production of the deoxyribonucleotide diphosphates, thus interfering with synthesis of deoxyribonucleotides and hampering cell cycle progression into the S phase. On the other hand, actinomycin D is widely used as a tool to inhibit, evaluate, and study transcription. Upon cell incubation (for 4 h) with EC in the presence of hydroxyurea (Fig. 2A, iii) or thymidine (Fig. 2A,
Fig.3. EC and EU detection in various tissues of animals. Mice were injected ip with 2 mg of EC or EU. Organs were harvested 5 or 24 h after injection. The organ sections were treated with Cy3-azide and DAPI, washed, and then imaged by fluorescence microscopy. (A1-A2) Distinct EC- and EU-generated signals in liver, colon, and ileum, but not kidney sections, detected 5 h after injection. (B1-B2) 24 h after injection, the EC-generated signal decreased sharply, while the EU-generated weakened signal still could be detected in liver, colon, and ileum. (C) The control results of various tissues harvested 5 h after injection.
5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135
iv), the intensity and subcellular pattern of EC stain were indistinguishable from those obtained after incubation with EC alone (Fig. 2A, ii). The results obtained with EC were also similar to those obtained with EU (Fig. 2B, ii–iv), demonstrating that EC did not incorporate into replicating DNA at substantial levels. To further demonstrate that EC could label cellular RNA, but not DNA, cells were treated with EC in the presence of actinomycin D at various concentrations. It was well documented that the presence of actinomycin D at low concentrations inhibited RNA polymerase I activity, predominantly responsible for transcription and processing of highly abundant rRNA in the nucleoli compartment of the nucleus [20]. Consistent with EC’s ability to incorporate and label nascent rRNA transcripts, the intense staining of nucleoli was abolished at the low concentration of actinomycin D (while leaving the EC signal in the rest of the cell unchanged; Fig. 2A, v), thus confirming that the original EC staining of nucleoli
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indeed represented the RNA polymerase I transcripts, most probably the pre-rRNA molecules. High concentrations of actinomycin D are known to also inhibit RNA polymerase II activity. Consistent with this, the EC staining was prevented throughout the whole cell (the effect similar to that observed with EU) upon exposure to actinomycin D at high concentrations (Fig. 2A, vi, and B, vi). Taken together, these results demonstrated that EC could incorporate into RNA transcripts produced by RNA polymerases I and II, but not into the nascent DNA. Labeling of nascent nucleic acids in cells using alkynyl-substituted purine nucleosides We next tested the labeling pattern in cells treated with alkynyl-substituted purine nucleoside analogues (EA), which turned out to be very different from that of alkynyl-substituted
Fig. 3. (continued)
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Fig. 3. (continued)
pyrimidine ribonucleoside analogue (EU and EC)-treated cells, producing pronounced staining not only of nuclei, but also of the cytoplasm (Supplementary Fig. S2, ii). Inhibition of DNA synthesis by hydroxyurea or thymidine, as well as inhibition of RNA synthesis by 2 lM actinomycin D, did not change this labeling pattern (Supplementary Fig. S2, iii–vi). Even when both DNA and RNA synthases were inhibited simultaneously by incubating cells with hydroxyurea and actinomycin D or with thymidine and actinomycin D combinations, the cellular staining pattern remained the same (Supplementary Fig. S2, vii and viii). We cannot explain this result at this point and expect that additional study of the alkynylsubstituted purine nucleosides metabolism would be required to provide an explanation. In any case, these results argued that alkynyl-substituted purine nucleoside analogues could not be used for specific detection of RNA or DNA synthesis in the cells. The use of EC in various cell types Whether EC could be used to monitor synthesis of nucleic acids in other cell types, different from HeLa cells (described above), was investigated. For this, six cell types (A549, C2C12, H1299, HLF, LLC, and HUVECs) were incubated with 1 mM EC for 4 h, then fixed by 4% paraformaldehyde in PBS, and subsequently stained with fluorescent Cy3-azide and DAPI. Results demonstrated that EC was readily incorporated into all tested cell types and could be used universally to monitor RNA synthesis (Supplementary Fig. S3).
tions were subsequently stained with fluorescent Cy3-azide and DAPI, washed, and then imaged by fluorescence microscopy. The labeling results of four different organs (liver, kidney, colon, and ileum) are summarized in Fig. 3. After the 5-h time point, no obvious EC or EU staining signal could be detected in kidney (Figs. 3A1, ii and 3A2, ii) or spleen (data not shown), although a former report demonstrated that kidney tubules and a large subset of the lymphocytes in spleen showed intensive EU staining [9]. Both EC and EU produced similar labeling patterns in liver, with very intense signal coming from hepatocytes (Fig. 3A1, i, and A2, i), consistent with hepatocytes showing strong EU staining in a prior report [9]. In colon, distinct EC-generated labeling signal was detected at the tips of villi (Fig. 3A1, iii), while the overall intensity of the EC-generated signal was lower than that originating from EU (compare Fig. 3A1, iii, and A2, iii). In ileum, EC produced strong signal at the tips of villi (Fig. 3A1, iv), while the EU-generated signal came from the base of villi (Fig. 3A2, iv). No EC-generated signal could be detected in any of four evaluated organs after 24 h (Fig. 3B1). Distinct signal still could be detected in liver, colon, and ileum of EU-injected animals (Fig. 3B2, i, iii, and iv) after 24 h; however, the strength of the signal was substantially weaker than after 5 h of injection. In ileum, the EU labeling shifted from the base of the villi toward their tips (compare Fig. 3A2, iv, and B2, iv). In summary these results demonstrate that EC was well suited to detecting RNA synthesis in proliferating tissues and that the metabolic rate of EC was faster than that of EU.
Monitoring RNA synthesis in animals using EC Conclusions To determine whether EC could be used to label RNA in animals, two 3-week-old mice were injected ip with 2 mg of EC in PBS. An uninjected littermate was used as a control. A parallel experiment was conducted with EU to compare the efficacy of labeling and the labeling patterns. Animals were euthanized and tissues collected and fixed at 5- and 24-h time points after injection. Paraffin sec-
We have demonstrated that 5-ethynylcytidine (EC), a novel cytidine analogue, could be used efficiently to monitor RNA synthesis in vitro and in vivo when coupled with Cy3-azide via a Cu(I)-catalyzed click reaction. EC demonstrated sensitivity comparable to that of EU and higher metabolic rate than EU. In contrast to
5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135
EC, alkynyl-substituted purine nucleosides failed to label specifically the newly transcribed RNA. We predict that EC will have applications for monitoring of RNA synthesis and assaying the turnover of transcripts both in vitro and in vivo. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 30870535 and 90913017), the Combination Project of Guangdong Province and the Ministry of Education (No. 2009B090300080 and 2011B090400478), the Introduced Innovative R&D Team Program of Guangdong Province (No. 201001Y0104789252), and the 863 Program of China (No. 2012AA022501). 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.2012.11.023. References [1] M. Uddin, G.G. Altmann, C.P. Leblond, Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine, J. Cell Biol. 98 (1984) 1619–1629. [2] D. Wansink, W. Schul, I. van der Kraan, B. van Steensel, R. van Driel, L. de Jong, Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus, J. Cell Biol. 122 (1993) 283– 293. [3] D. Cmarko, P.J. Verschure, T.E. Martin, M.E. Dahmus, S. Krause, X.D. Fu, R. van Driel, S. Fakan, Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection, Mol. Biol. Cell 10 (1999) 211–223. [4] X. Wei, S. Somanathan, J. Samarabandu, R. Berezney, Three-dimensional visualization of transcription sites and their association with splicing factorrich nuclear speckles, J. Cell Biol. 146 (1999) 543–558. [5] G. Haukenes, A.M. Szilvay, K.A. Brokstad, A. Kanestrøm, K.H. Kalland, Labeling of RNA transcripts of eukaryotic cells in culture with BrUTP using a liposome transfection reagent (DOTAP), Biotechniques 22 (1997) 308–312. [6] N. Sadoni, D. Zink, Nascent RNA synthesis in the context of chromatin architecture, Chromosome Res. 12 (2004) 439–451.
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