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A Covalent Approach for Sitespecific RNA Labeling in Mammalian Cells

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A Covalent Approach for Sitespecific RNA Labeling in Mammalian Cells Angewandte Chemie International Edition:DOI:10.1002/anie.201410433 RNA Labeling Very Important Paper German Edition:DOI:10.1002/ange.201410433 ACovalent Approach for Site-Specific RNALabeling in Mammalian Cells** Fahui Li, Jianshu Dong, Xiaosong Hu, Weimin Gong,Jiasong Li, Jing Shen, Huifang Tian, and Jiangyun Wang* Abstract: Advances in RNAresearch and RNAnanotechnol- approaches,[2] bioorthogonal chemistry[3] relies upon the ogy depend on the ability to manipulate and probe RNAwith specific and covalent attachment of probe molecules to the high precision through chemical approaches,both in vitro and biomacromolecule of interest (BOI). As aresult, it offers in mammalian cells.However,covalent RNAlabeling methods several advantages:1)the affinity can be considered to be with scope and versatility comparable to those of current infinity,therefore stringent wash conditions can be applied to protein labeling strategies are underdeveloped. Amethod is rigorously purify the BOI;2)the high affinity allows for the reported for the site- and sequence-specific covalent labeling of visualization of low-abundance BOIs,which is particularly RNAs in mammalian cells by using tRNAIle2-agmatidine important for non-coding RNAs;[1a] 3) this method is synthetase (Tias) and click chemistry.The crystal structure of extremely versatile;probe molecules harboring fluorescence, Tias in complex with an azide-bearing agmatine analogue was NMR, IR, or EPR functional groups can be conveniently solved to unravel the structural basis for Tias/substrate conjugated to the BOI.[3] Because of these unique features, recognition. The unique RNAsequence specificity and plastic bioorthogonal chemistry has been successfully employed in Tias/substrate recognition enable the site-specific transfer of protein and glycan functional studies to visualize their azide/alkyne groups to an RNAmolecule of interest in vitro expression, localization, and interaction partners in mamma- and in mammalian cells.Subsequent click chemistry reactions lian cells.[3] facilitate the versatile labeling,functionalization, and visual- While engineering of the translational[4] or post-transla- ization of target RNA. tional machinery[5] in combination with bioorthogonal chemistry has enabled protein labeling with unprecedented Ithas recently emerged that RNAhas functions as diverse precision and versatility,[6] comparable methods for the site- and important as those of proteins.[1] To unravel the dynamic and sequence-specific tagging of RNAs in mammalian cells is trafficking,localization, and interactions of RNAinliving currently underdeveloped.[7] To achieve this,weneed to cells,numerous methods have been developed for the non- engineer unique components of the post-transcriptional covalent labeling of RNAs in mammalian cells,and dramatic machinery and employ bioorthogonal chemistry.First, an progress has been made.[2] Compared to the noncovalent RNA-modifying enzyme must recognize aspecific site in aunique RNAsequence in the transcriptome.Second, this [*] F. H. Li,[+] J. S. Dong,[+] W. M. Gong, J. S. Li, J. Y. Wang enzyme must be able to transfer asmall molecule bearing Laboratory of RNA Biology and Laboratory of Quantum Biophysics aunique chemical handle (such as an azide or alkyne group) Institute of Biophysics, Chinese Academy of Sciences to the specific site.Subsequent covalent labeling through 15 Datun Road, Chaoyang District, Beijing, 100101 (China) bioorthogonal chemistry then leads to the site-specific con- E-mail:[email protected] jugation of reporter groups,thus enabling fluorescence, J. Y. Wang NMR, EPR, or IR spectroscopy measurements (Figure 1). Beijing National Laboratory for Molecular Sciences (BNLMS) To search for apost-transcriptional modification module chinaXiv:201605.01192v1 Institute of Chemistry,Chinese Academy of Sciences Beijing,100190 (China) that can be used for precise RNAlabeling in mammalian cells, X. S. Hu[+] we turned to the tRNA-modifying enzymes,which are known [8] Department of Chemistry,School of Chemistry to catalyze around 100 kinds of tRNAmodification. Faithful ChemicalEngineering and Life Sciences translation of the genetic code relies on the chemical Wuhan University of Technology,Wuhan 430070 (China) modification of tRNA,[9] especially at the wobble position 34 J. Shen, H. F. Tian of the anticodon.[10] Many tRNA-modifying enzymes acting The Laboratory of Carcinogenesis and Translational Research on this position are found only in specific domains,thus (Ministry of Education) implying that they may have evolved independently.[11] Core Laboratory,Peking University School of Oncology Notably,the C34 position of the AUA-decoding tRNAIle2 is Beijing Cancer Hospital & Institute, Beijing, 100142 (China) modified with lysine by the enzyme lysidine synthetase (Tils) [+]These authors contributed equally to this work. in bacteria,[12] but with agmatine (AGM, 1;Scheme 1) by the [**] We gratefully acknowledge the Major State Basic Research Program enzyme tRNAIle2-agmatidine synthetase (Tias) in arch- of China (2015CB856203), the CAS grant (KJZD-EW-L01), and the [13,14] Ile NationalScience Foundation of China (91440116, 21325211, aea. In eukaryotes,tRNA bearing pseudouridine (Y) 91313301,31370016,21102172). We thank YanTeng for help with or inosine (I) at the wobble position decodes the AUA [9,15] fluorescence imaging. codon. As shown previously, Archaeoglobus fulgidus (Af) Ile2 Supporting information for this article is available on the WWW Tias recognition of tRNA requires seven nucleotides:G1, under http://dx.doi.org/10.1002/anie.201410433. G2, C34, U36, A37, C71, and C72 (Figure S13A in the Angew.Chem. Int.Ed. 2015, 54,4597 –4602 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim 4597 Angewandte. Communications Figure 1. A) In the presence of ATP, Tias catalyzes the conjugation of agmatine to C34 of tRNAIle2,thereby resulting in the formation of agmatidine. B) Labeling of an RNA of interest (ROI). Tias facilitates the site-specific transfer of an agmatine analogue bearing an azide group to the ROI-tRNAIle2 fusion RNA. Subsequent strain-promoted azide–alkyne cycloaddition allows the conjuration of biophysical probes. Scheme 1. Structure of agmatine (AGM) and agmatineanalogues. Figure 2. A) Acid–urea PAGE with tRNAIle2 modified by using Tias and [13,14] AGM analogues. The gel was stained by ethidium bromide (EB). Supporting Information). Since neither Tias nor tRNA B) While Tias catalyzes the efficient conjugation of AGM (1)and AGN bearing this set of identity elements is present in mammalian (2)toAftRNAIle2,the reaction does not occur for the Af tRNAIle2 C34U cells (Figure S13),[16] we envisioned that the introduction of mutant, thus indicating that the small-molecule probes are transferred Tias and archaeal tRNAIle2 into mammalian cells could specifically to the C34 position. The reaction mixtures were analyzed facilitate the sequence- and site-specific labeling of RNAif by acid–urea PAGE. MALDI-TOF analysesare shown for unlabeled Af Ile2 Ile2 Ile2 Tias can transfer small molecule probes bearing azide or tRNA (C), Af tRNA modified with 1 (D), Af tRNA modified with 2 (E), and Af tRNAIle2 modified with 3 (F). alkyne functional groups to an RNAofinterest. As afirst step to developing acovalent method for labeling RNAs,wesynthesized three AGManalogues: N-(4- compound 4.The much bulkier carbamate and alkyne aminobutyl)-2-azidoacetamide (AGN, 2), 2-propynylamine functional groups may result in inefficient recognition of (3), and (but-3-yn-1-yl)(4-aminobutyl)carbamate (4; compound 4 by Tias.Wetherefore choose compounds 2 and 3 Scheme 1). Modification of Af tRNAIle2 was performed with for further investigation. Tias (8 mm), Af tRNAIle2 (1 mm), Tris-HCl (100 mm), pH 8.0, We then investigated whether Tias catalyzes the specific Ile2 KCl (10 mm), MgCl2 (5 mm), DTT (5 mm), ATP(1mm), and modification of nucleotide C34 of Af tRNA .While Tias substrates 1, 2, 3, or 4(1mm). AGM(1) was used as apositive catalyzed the efficient ligation of compounds 1 and 2 to Af control. Themodified and unmodified RNAs were then tRNAIle2,this activity was abolished for the C34U mutant separated by 6.5% acid–urea polyacrylamide gel electro- (Figure 2B). These results indicate that Tias can target both phoresis (PAGE), as previously described.[17] When tRNAIle2 the natural substrate AGMand the unnatural AGMana- was modified, the modified RNAexhibited lower mobility logues specifically to nucleotide C34 of Af tRNAIle2.To (Figure 2A). To our delight, we found that in the presence of further demonstrate that compounds 2 and 3 conjugate substrates 2, 3,or4,tRNAIle2 was covalently modified, thus precisely with nucleotide C34 of Af tRNAIle2,wedigested resulting in lower mobility in the acid–urea polyacrylamide Af tRNAIle2 with RNase T1 (Figure S15)[18] and then analyzed gel. Perhaps owing to the lower molecular weight of the RNAfragment containing the anticodon loop by compound 3,tRNAIle2 modified with compound 3 exhibited MALDI-TOF. Theunmodified tRNAIle2 fragment gave an higher mobility than tRNAIle2 modified with compound 2.In observed average mass of 3184.08 Da, which closely matches the presence of compound 4,two major bands were present, the calculated mass of 3184.4 Da (Figure 2). After AGMor2- thus indicating that tRNAIle2 was only partially modified with propynylamine modification, the tRNAIle2 fragment gave 4598 www.angewandte.org 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Angew.Chem.Int. Ed. 2015, 54,4597 –4602 Angewandte Chemie observed average masses of 3295.3 and 3220.3 Da, respec-
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