Analytical Biochemistry 434 (2013) 128–135

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Analytical Biochemistry

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5-Ethynylcytidine as a new agent for detecting RNA synthesis in live cellsby ‘‘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, Chinab Graduate University of the Chinese Academy of Sciences, Beijing 100049, Chinac Guangzhou RiboBio Co., Ltd., Guangzhou Science Park, Guangzhou 510663, Chinad 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 a b s t r a c t

Article history:Received 29 September 2012Received in revised form 22 November 2012Accepted 27 November 2012Available online 3 December 2012

Keywords:"Click" chemistryEU5-EthynylcytidineRNA labeling

0003-2697/$ - see front matter Crown Copyright � 2http://dx.doi.org/10.1016/j.ab.2012.11.023

⇑ Corresponding author. Fax: +86 20 32290137.E-mail address: Zhang_biliang@gibh.org (B. Zhang

1 These authors contributed equally to this work.2 Abbreviations used: EC, 5-ethynylcytidine; EA

5-bromouridine; EU, 5-ethynyluridine; DMEM, DulbeccFBS, fetal bovine serum; DAPI, 40 ,6-diamidino-2-phennetic resonance; Cy3-azide, 2-[3-(1,3-dihydro-1,1-dbenz[e]indol-2-ylidene)propenyl]-3,3-dimethyl-1-ethyldichloromethane; DMSO, dimethyl sulfoxide; PBS, pintraperitoneally.

Detection of RNA synthesis in cells to measure the rate of total transcription is an important experimentaltechnique. To screen the best nucleoside analogue for labeling RNA synthesis, a series of alkyne-modifiednucleoside analogues, including 5-ethynylcytidine (EC) and 8-ethynyladenosine (EA), were successfullysynthesized by the Sonogashira coupling reaction. The synthesis of RNA or DNA was assayed based onthe biosynthetic incorporation of these analogues into newly transcribed RNA or replicating DNA. Ana-logue-labeled cellular RNA or DNA was detected quickly and with high sensitivity via ‘‘click’’ chemistrywith fluorescent azides, followed by fluorescence microscopic imaging. The results showed that EC wasefficiently incorporated into RNA, but not into DNA, in seven cell lines, as also previously shown for 5-eth-ynyluridine (EU). Moreover, EC was able to assay transcription rates of various tissues in animals and therate of metabolism of EC was much faster than that of EU.

Crown Copyright � 2012 Published by Elsevier Inc. All rights reserved.

Detection and quantification of RNA synthesis in cells is awidely used technique to monitor cell viability, health, and metab-olism rate. Until recently, two approaches for direct monitoring ofRNA synthesis have been used.

The first method relies on incorporation of the radioisotope-labeled nucleosides, e.g., [3H]uridine, followed by tissue autoradi-ography [1]. Such approach is cumbersome and slow and has lowsensitivity, requiring exposure times of weeks to months underconditions of complete exclusion of external light. The need touse radioactive materials also requires a higher level of experimen-tal caution and a special laboratory setup. Therefore, many clinicaland research laboratories prefer to avoid this technique [1].

The second method was developed as a nonradioactive alterna-tive and engages incorporation into nascent RNA of uridine chem-ical analogues, such as 5-bromouridine (BrU)2, which can then beimmunodetected using specific antibodies. BrU can be introduced

012 Published by Elsevier Inc. All r

).

, 8-ethynyladenosine; BrU,o’s modified Eagle’s medium;ylindole; NMR, nuclear mag-imethyl-3-(6-azidohexyl)-2H--3H-indolium bromide; DCM,hosphate-buffered saline; ip,

to the cells in the form of a nucleoside or as a 50-triphosphate nucle-otide. Since the cellular membrane is impermeable to 5-bromouri-dine triphosphate, various techniques have been used to deliverthis 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 canbe taken up by cells spontaneously, where it is converted into 50-mono-, 50-di-, and 50-triphosphate derivatives by cellular kinasesand subsequently incorporated into the newly synthesized RNAtranscripts [8]. Although the use of BrU is safer and more convenientthan that of [3H]uridine, it poses significant limitation, since the BrUantibody is large and, hence, does not penetrate the tissues. There-fore, this approach has very limited use in whole animals or intacttissues.

Most recently, a new method for RNA synthesis monitoring hasbeen developed, which involves the incorporation of 5-ethynyluri-dine (EU), a uridine analogue, into cellular RNA and subsequentreaction of EU with a fluorescent azide via ‘‘click’’ chemistry [9].This approach offers big promise in the detection and quantifica-tion of RNA synthesis, since the reaction is highly reliable, efficient,and selective. Importantly, azides and alkynes are bio-orthogonalmolecules and are compatible with a wide range of solvents,including water. Furthermore, fluorescent azides are very smalland are just 1/500 the size of an antibody. Thus fluorescent azidesdemonstrate very high diffusion rate and ability to penetrate intactanimal tissues effectively [9–11]. All these advantages grant the

ights reserved.

5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135 129

EU-based RNA synthesis monitoring assays higher sensitivity andshorter procedural timing [12,13].

To further improve the efficiency, speed, and sensitivity of RNAsynthesis detection and quantification, we have synthesized andtested a series of alkyne-modified nucleoside analogues. Uponexamination of new analogue performance both in vitro andin vivo, we propose a novel, more advanced approach for monitor-ing of RNA synthesis in cell culture and animals.

Materials and methods

HeLa (human epithelial cervical cancer cell line), C2C12 (mousemuscle myoblast cell line), HLF (human embryonic lung fibroblastcell line), and LLC (mouse Lewis lung cancer cell line) cells weregrown in Dulbecco’s modified Eagle’s medium (DMEM). A549 (hu-man lung cancer cell line) and H1299 (human non-small-cell lungcarcinoma cell line) cells were maintained in RPMI 1640 medium.HUVECs (human umbilical vein endothelial cells) were maintainedin EGM-2 medium (Clonetics). All media were supplemented with10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Cells weregrown in an atmosphere of 5% CO2 at 37 �C. All cell lines werekindly provided by Duanqing Pei’s laboratory at the GuangzhouInstitute of Biomedicine and Health (Guangzhou, China).

All of the basic chemicals, such as hydroxyurea, thymidine, acti-nomycin D, CuSO4, ascorbic acid, paraformaldehyde, glycine, and40,6-diamidino-2-phenylindole (DAPI), as well as the syntheticcompounds, such as 2-(2-anilinovinyl)-1-ethyl-3,3-dimethyl-3H-indolium 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 Sonogashiracoupling reaction

Copper(I) iodide (4 mM), anhydrous triethylamine (20 ml), tri-methylsilylacetylene (20 mM), and tetrakis(triphenylphos-phine)palladium(0) (2 mM) were added to a degassed solution ofthe desired nucleoside (10 mM) in anhydrous dimethyl formamide(100 ml), and the mixture was left to stir at temperature from 25 to50 �C. After 6 h the solvent was removed in vacuum and the resi-due dissolved in methanol (100 ml) and reacted with potassiumcarbonate (15 mM) to remove the trimethylsilyl group, yieldingethynyl-modified nucleoside analogues. Ethynyl-modified nucleo-side analogues were then purified by flash chromatography onsilica.

5-EthynyluridineEU was synthesized from 5-iodouridine and trimethylsilylacet-

ylene using the Sonogashira coupling reaction according to generalprocedure 1 (above). Purification by wet flash column chromatog-raphy eluting with methanol in dichloromethane (15–25%) yieldedEU 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).

5-Ethynylcytidine (EC)EC was synthesized from 5-iodocytidine and trimethylsilylacet-

ylene using the Sonogashira coupling reaction according to generalprocedure 1 (above). Purification by wet flash column chromatog-raphy (silica preequilibrated with 1% Et3N) eluting with methanolin dichloromethane (15–30%) yielded EC as a light yellow powder(1.28 g, 48%). EC analysis: light yellow powder, molecular weight267.24; ES-API LC/MS M = 267.09 (m/z), [M+H]+ = 268.1,[M+Na]+ = 290.1, [2M+Na]+ = 557.2; 1H 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);13C 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 trimethylsilyl-

acetylene using the Sonogashira coupling reaction according togeneral procedure 1 (above). Purification by wet flash columnchromatography (silica preequilibrated with 1% Et3N) eluting withmethanol in dichloromethane (20–35%) yielded EA as a light yel-low powder (1.48 g, 51%). EA analysis: light yellow powder, molec-ular 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, H-40), 3.664–3.527 (dm, 2H, H-50); 13C NMR (DMSO-d6, 500 MHz): d156.263 (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)-2H-benz[e]indol-2-ylidene)propenyl]-3,3-dimethyl-1-ethyl-3H-indoliumbromide (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 pyr-idine (25 ml) and stirred for 30 min. 1,1,2-Trimethyl-3-(6-azi-dohexyl)-1H-benzo[e]indolium bromide (5.4 g, 13 mmol) wasadded and the mixture was heated at 110 �C for 10 h. After the sol-vent was evaporated, the residue was dissolved in 100 ml ofdichloromethane (DCM), washed with water, and dried over so-dium sulfate. After the solvent was evaporated, the residue waspurified by silica-gel column chromatography with DCM/methanol(15:1) to yield the product as a purple solid (2.0 g, yield 28.0%). 1HNMR (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 forC35H42N5 [M]+: 532.3440. Found: 532.3444.

RNA labeling using alkyne-modified nucleoside analogues in culturedcells

HeLa cells were grown on glass coverslips in DMEM supple-mented with 10% FBS. EU and EC were added to the complete cul-ture medium from a 100 mM stock in H2O. EA was added to thecomplete culture medium from a 200 mM stock in DMSO.

130 5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135

After exposure to nucleoside analogues for 4 h, all cells werewashed with PBS and fixed with 4% paraformaldehyde in PBS for30 min at room temperature, then incubated with glycine at theconcentration of 2 mg/ml in PBS to stop the fixing action of para-formaldehyde, and finally permeabilized by 0.2% Triton X-100 inPBS. In the case of EC, the analogue was added to the complete cul-ture medium from a 100 mM stock in H2O and rinsed once withPBS, followed by click staining using Cy3-azide.

To evaluate the labeling effect on DNA synthesis when it wasinhibited, 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 mMhydroxyurea or 2 mM thymidine. To examine the labeling effecton RNA synthesis when it was inhibited, HeLa cells were grownfor 4 h in complete medium with 1 mM EU, 1 mM EC, or 5 mMEA, with or without actinomycin D (100 nM or 2 lM). The cellswere then rinsed, fixed, and processed for azide coupling and DAPIstaining. To evaluate the synchronous inhibition of DNA and RNAsynthesis, cells were incubated for 4 h with 5 mM EA in the pres-ence or absence of 10 mM hydroxyurea and 2 lM actinomycin Dor 2 mM thymidine and 2 lM actinomycin D.

Fig.1. Label on nascent RNA in cells using alkyne-modified nucleoside analogues. (A)analogues incorporated into cellular RNA. The terminal alkyne groups of bases readilyreaction (‘‘click’’ chemistry). (B1–B4) Structures of the fluorescent azide and the alkyne-

Alkyne-modified nucleoside analogue detection by click chemistry

All the steps of alkyne-modified nucleoside analogue detectionwere performed at room temperature. The fixed cells were rinsedwith PBS and stained for 30 min at room temperature with100 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 al-kyne-modified nucleoside analogues, cells were washed two timeswith methanol, once with PBS, and then stained with DAPI. Theresulting labeled and stained cells were imaged by fluorescencemicroscopy.

EC treatment of C2C12, HLF, LLC, A549, H1299, and HUVEC cells

C2C12, HLF, and LLC cells were grown on glass coverslips inDMEM supplemented with 10% FBS. A549 and H1299 cells weregrown on glass coverslips in 1640 supplemented with 10% FBS.HUVECs were grown on glass coverslips in EGM-2 supplementedwith 5% FBS. The cells were then incubated with 1 mM EC for

Schematic diagram of the click reaction for detecting alkyne-modified nucleosidereact with an organic fluorescent azide via a Cu(I)-catalyzed [3 + 2] cycloadditionmodified nucleoside analogues used in this study.

5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135 131

4 h, rinsed, fixed, and stained with Cy3-azide and DAPI. The result-ing cells were imaged by fluorescence microscopy.

EC and EU labeling of mouse tissues

The 3-week-old mice were injected intraperitoneally (ip) with2 mg EC or EU in 0.1 ml PBS. A control littermate mouse was in-jected ip with 0.1 ml PBS. The mouse liver, kidney, spleen, colon,and ileum were harvested 5 or 24 h after injection. Pieces of theharvested tissues were embedded in paraffin and sectioned. Afterparaffin removal and rehydration, the sections were stained withCy3-azide and DAPI using the same protocol as for cultured cells(above). Upon staining, the samples were mounted for fluores-cence 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

Fig.2. Monitoring EC incorporation into nascent RNA in cultured HeLa cells using fluothymidine, and 100 nM or 2 lM actinomycin D in the presence of 1 mM EC or EU for 4fluorescence microscopy. (A) EC. (B) EU.

alkyne-modified nucleoside analogues including EU, EC, and EA(Fig. 1B2–B4) using the Sonogashira coupling reaction as previ-ously described [14–19]. To be used as a detecting agent for theseanalogues, we synthesized a fluorescent azide derivative, Cy3-azide (Fig. 1B1). HeLa cells were cultured with 1 mM EU, 1 mMEC, and 5 mM EA for 4 h, respectively, and then fixed and subse-quently stained with Cy3-azide and DAPI. Last, the cells were im-aged by fluorescence microscopy at excitation wavelengths of488 and 543 nm for DAPI and Cy3, respectively. The first row ofeach figure shows the newly nascent RNA transcripts or newly rep-licated 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 andblue. Untreated HeLa cells were used as a control.

The labeling pattern with EC was similar to that obtained withEU (Supplementary Fig. S1, ii and iii). All cells were uniformly la-beled, with EC-labeled cells also producing strong signals fornucleoli, the cellular compartment responsible for transcriptionof most abundant ribosomal RNA. Upon incubation with EA, thecells showed strong staining in both nuclei and cytoplasm (Supple-mentary Fig. S1, iv).

rescence microscopy. HeLa cells were incubated with 10 mM hydroxyurea, 2 mMh. The cells were fixed, treated with Cy3-azide and DAPI, washed, and imaged by

132 5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135

EC labels cellular RNA, but not DNA

To exert the RNA labeling effect, EC needs to enter the ribonu-cleoside salvage pathway and be metabolized into its triphosphateform in three subsequent phosphorylation steps: EC ? ECM-P ? ECDP ? ECTP. Ribonucleotide reductase is the enzyme thatcatalyzes the formation of deoxyribonucleotide diphosphates fromribonucleotide diphosphates. If ECDP were a substrate for ribonu-cleotide reductase, the intermediate EdCDP would be phosphory-lated to its triphosphate form (EdCTP), which, in its turn, wouldbe used in the synthesis of DNA as well. Such outcome would limitthe utility of the EC-based labeling procedure, since both the nas-

Fig.3. EC and EU detection in various tissues of animals. Mice were injected ip with 2 mgtreated with Cy3-azide and DAPI, washed, and then imaged by fluorescence microscopykidney sections, detected 5 h after injection. (B1-B2) 24 h after injection, the EC-generatdetected in liver, colon, and ileum. (C) The control results of various tissues harvested 5

cent replicating DNA and the transcribed RNA would be labeled,thus making it difficult to distinguish and differentiate the twoprocesses. To exclude such possibility, we next tested whetheraddition of EC to cells in culture would result in DNA labeling.

Hydroxyurea and thymidine are the two well-characterizedinhibitors of ribonucleotide reductase. Upon exposure to cells, theycause decreased production of the deoxyribonucleotide diphos-phates, thus interfering with synthesis of deoxyribonucleotidesand hampering cell cycle progression into the S phase. On the otherhand, actinomycin D is widely used as a tool to inhibit, evaluate,and study transcription. Upon cell incubation (for 4 h) with EC inthe presence of hydroxyurea (Fig. 2A, iii) or thymidine (Fig. 2A,

of EC or EU. Organs were harvested 5 or 24 h after injection. The organ sections were. (A1-A2) Distinct EC- and EU-generated signals in liver, colon, and ileum, but not

ed signal decreased sharply, while the EU-generated weakened signal still could beh after injection.

5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135 133

iv), the intensity and subcellular pattern of EC stain were indistin-guishable from those obtained after incubation with EC alone(Fig. 2A, ii). The results obtained with EC were also similar to thoseobtained with EU (Fig. 2B, ii–iv), demonstrating that EC did notincorporate into replicating DNA at substantial levels.

To further demonstrate that EC could label cellular RNA, but notDNA, cells were treated with EC in the presence of actinomycin Dat various concentrations. It was well documented that the pres-ence of actinomycin D at low concentrations inhibited RNA poly-merase I activity, predominantly responsible for transcriptionand processing of highly abundant rRNA in the nucleoli compart-ment of the nucleus [20]. Consistent with EC’s ability to incorpo-rate and label nascent rRNA transcripts, the intense staining ofnucleoli 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

Fig. 3. (con

indeed represented the RNA polymerase I transcripts, most proba-bly the pre-rRNA molecules. High concentrations of actinomycin Dare known to also inhibit RNA polymerase II activity. Consistentwith this, the EC staining was prevented throughout the whole cell(the effect similar to that observed with EU) upon exposure to acti-nomycin D at high concentrations (Fig. 2A, vi, and B, vi). Taken to-gether, these results demonstrated that EC could incorporate intoRNA transcripts produced by RNA polymerases I and II, but not intothe nascent DNA.

Labeling of nascent nucleic acids in cells using alkynyl-substitutedpurine nucleosides

We next tested the labeling pattern in cells treated with alky-nyl-substituted purine nucleoside analogues (EA), which turnedout to be very different from that of alkynyl-substituted

tinued)

Fig. 3. (continued)

134 5-Ethynylcytidine for RNA Labeling in live cells / D. Qu et al. / Anal. Biochem. 434 (2013) 128–135

pyrimidine ribonucleoside analogue (EU and EC)-treated cells, pro-ducing pronounced staining not only of nuclei, but also of the cyto-plasm (Supplementary Fig. S2, ii). Inhibition of DNA synthesis byhydroxyurea or thymidine, as well as inhibition of RNA synthesisby 2 lM actinomycin D, did not change this labeling pattern (Sup-plementary Fig. S2, iii–vi). Even when both DNA and RNA syn-thases were inhibited simultaneously by incubating cells withhydroxyurea and actinomycin D or with thymidine and actinomy-cin D combinations, the cellular staining pattern remained thesame (Supplementary Fig. S2, vii and viii). We cannot explain thisresult at this point and expect that additional study of the alkynyl-substituted purine nucleosides metabolism would be required toprovide an explanation. In any case, these results argued that alky-nyl-substituted purine nucleoside analogues could not be used forspecific 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 acidsin other cell types, different from HeLa cells (described above), wasinvestigated. For this, six cell types (A549, C2C12, H1299, HLF, LLC,and HUVECs) were incubated with 1 mM EC for 4 h, then fixed by4% paraformaldehyde in PBS, and subsequently stained with fluo-rescent Cy3-azide and DAPI. Results demonstrated that EC wasreadily incorporated into all tested cell types and could be useduniversally to monitor RNA synthesis (Supplementary Fig. S3).

Monitoring RNA synthesis in animals using EC

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. Anuninjected littermate was used as a control. A parallel experimentwas conducted with EU to compare the efficacy of labeling and thelabeling patterns. Animals were euthanized and tissues collectedand fixed at 5- and 24-h time points after injection. Paraffin sec-

tions were subsequently stained with fluorescent Cy3-azide andDAPI, washed, and then imaged by fluorescence microscopy. Thelabeling results of four different organs (liver, kidney, colon, andileum) are summarized in Fig. 3.

After the 5-h time point, no obvious EC or EU staining signalcould be detected in kidney (Figs. 3A1, ii and 3A2, ii) or spleen(data not shown), although a former report demonstrated that kid-ney tubules and a large subset of the lymphocytes in spleenshowed intensive EU staining [9]. Both EC and EU produced similarlabeling patterns in liver, with very intense signal coming fromhepatocytes (Fig. 3A1, i, and A2, i), consistent with hepatocytesshowing strong EU staining in a prior report [9]. In colon, distinctEC-generated labeling signal was detected at the tips of villi(Fig. 3A1, iii), while the overall intensity of the EC-generated signalwas lower than that originating from EU (compare Fig. 3A1, iii, andA2, iii). In ileum, EC produced strong signal at the tips of villi(Fig. 3A1, iv), while the EU-generated signal came from the baseof villi (Fig. 3A2, iv).

No EC-generated signal could be detected in any of four evalu-ated organs after 24 h (Fig. 3B1). Distinct signal still could be de-tected 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 sub-stantially weaker than after 5 h of injection. In ileum, the EU label-ing shifted from the base of the villi toward their tips (compareFig. 3A2, iv, and B2, iv). In summary these results demonstrate thatEC was well suited to detecting RNA synthesis in proliferating tis-sues and that the metabolic rate of EC was faster than that of EU.

Conclusions

We have demonstrated that 5-ethynylcytidine (EC), a novelcytidine analogue, could be used efficiently to monitor RNA syn-thesis in vitro and in vivo when coupled with Cy3-azide via aCu(I)-catalyzed click reaction. EC demonstrated sensitivity compa-rable 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) 128–135 135

EC, alkynyl-substituted purine nucleosides failed to label specifi-cally the newly transcribed RNA. We predict that EC will haveapplications for monitoring of RNA synthesis and assaying theturnover of transcripts both in vitro and in vivo.

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (No. 30870535 and 90913017), the Combina-tion Project of Guangdong Province and the Ministry of Education(No. 2009B090300080 and 2011B090400478), the IntroducedInnovative 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, inthe online version, at http://dx.doi.org/10.1016/j.ab.2012.11.023.

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