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 article info 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 abstract 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. Ana- logue-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-eth- ynyluridine (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 metab- olism rate. Until recently, two approaches for direct monitoring of RNA synthesis have been used. The first method relies on incorporation of the radioisotope- labeled nucleosides, e.g., [ 3 H]uridine, followed by tissue autoradi- ography [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 experimen- tal 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 alterna- tive and engages incorporation into nascent RNA of uridine chem- ical analogues, such as 5-bromouridine (BrU) 2 , which can then be immunodetected using specific antibodies. BrU can be introduced to the cells in the form of a nucleoside or as a 5 0 -triphosphate nucle- otide. Since the cellular membrane is impermeable to 5-bromouri- dine 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 5 0 - mono-, 5 0 -di-, and 5 0 -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 [ 3 H]uridine, it poses significant limitation, since the BrU antibody is large and, hence, does not penetrate the tissues. There- fore, 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-ethynyluri- dine (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 quantifica- tion 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 ⇑ 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, 8-ethynyladenosine; BrU, 5-bromouridine; EU, 5-ethynyluridine; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; DAPI, 4 0 ,6-diamidino-2-phenylindole; NMR, nuclear mag- netic resonance; Cy3-azide, 2-[3-(1,3-dihydro-1,1-dimethyl-3-(6-azidohexyl)-2H- benz[e]indol-2-ylidene)propenyl]-3,3-dimethyl-1-ethyl-3H-indolium bromide; DCM, dichloromethane; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; ip, intraperitoneally. Analytical Biochemistry 434 (2013) 128–135 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio