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 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 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 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 (Y) 91313301,31370016,21102172). We thank YanTeng for help with or (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

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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- tively,which match the calculated masses of 3295.5 and 3220.3 Da. After AGNmodification, the tRNAIle2 fragment gave an observed average mass of 3311.2 Da, while the calculated mass is 3337.5 Da. This can be explained by the loss of two nitrogen atoms and the addition of two hydrogen atoms during the laser irradiation used for MALDI-TOF, as previous observed.[19] Taken together,these results demon- strate that Tias can facilitate the conjugation of azide/alkyne- bearing AGManalogues 2 and 3 to nucleotide C34 in Af tRNAIle2. To unravel the molecular basis for AGNrecognition by Tias,wesolved the structure of Tias with AGNbound to its active site.The main chain carbonyl group of Asp193 and the side chain of Asn194 form two hydrogen bonds with the azide group of AGN, while the guanidine group of Arg217 forms ahydrogen bond with the carbonyl group of AGN(Figure 3).

Figure 4. A) Structure of BCN-FITC. B) AGN-modified tRNAIle2 was visualized through strain-promoted click reaction with BCN-FITC. The bottom panel (EB staining) indicates that equivalent amounts of tRNAIle2 with and without AGN modification were loaded. C) The structure of Sulfo-Cy5-azide. D) tRNAIle2 modified with 2-propynylamine was visualized through CuI-catalyzed click reaction with Sulfo-Cy5- azide. The bottom panel (EB staining) shows that equivalent amounts of tRNAIle2 with and without 2-propynylamine modifications were loaded.

These results indicate that BCN-FITC reacts specifically with the azide group present on tRNAIle2.Under similar conditions and in the presence of CuI catalyst (1 mm), Sulfo-Cy5-azide reacts specifically with tRNAIle2 modified with 2-ropynyl- amine (3), but not with tRNAIle2 without the modification Figure 3. A) Overall structure of Tias in complex with AGN. The overall (Figure 4C and 4D). structure of Tias resembles atennis racket. The AGN binding site is highlighted by ared box. B) Structure of the AGN binding site of Tias. Once we had demonstrated that Tias can facilitate the The Fo-Fcelectron density map of AGN was contoured at 2.7 s. site-specific incorporation of azide and alkyne groups into Nitrogen atoms are shown in blue, oxygen atoms in red, and carbon tRNAIle2,wethen asked whether this strategy could be used to atoms in yellow. label any RNAofinterest (ROI). Through in vitro run-off transcription, we synthesized tRNAIle2 harboring 3’ and 5’ extensions (termed tRNAIle2-3-5, Figure 5A). We then incu- Meanwhile,the side chain of Val203 stabilizes the carbon bated tRNAIle2-3-5 with Tias in the presence or absence of chain of agmatine through hydrophobic interactions.Similar AGN. After removing excess AGN, BCN-FITC was added. hydrophobic interactions may also be needed to facilitate Tias Acid–urea PAGE was then used to analyze the reaction. Only recognition of 2-propynylamine.Since Tias can also use in the presence of AGNdid BCN-FITC selectively label compounds 3 and 4 as substrates,although these are either tRNAIle2-3-5 (Figure 5B), thus indicating that Tias can much smaller or much bulkier than compound 2,itappears catalyze the conjugation of AGNtotRNAIle2-3-5 in the that Tias has asomewhat plastic binding pocket for primary presence of 3’ and 5’ extensions.This result is consistent with amine substrates.Weare currently testing whether Tias can previous reports that the agmatidine modification occurs also use primary amines bearing cyclopropene groups as early in the tRNAmaturation process to ensure high-fidelity substrates,which may facilitate photoclick reactions or translation.[13–14] inverse electron demand Diels–Alder reactions.[20] We then asked whether our method can be used to label We then tested whether Tias can facilitate RNAfluores- aspecific RNAinthe mammalian cell transcriptome.293T cence labeling through bioorthogonal chemistry.tRNAIle2 cells expressing Tias with or without tRNAIle2-5S fusion RNA with or without AGN(2)modification was incubated with expression were grown in medium containing AGN, followed BCN-FITC and then analyzed by 6.5%acid–urea PAGE, by cell lysis.Subsequently,total cellular RNAwas incubated followed by fluorescent imaging.tRNAIle2 modified with with BCN-Cy5. In the absence of tRNAIle2-5S expression, AGNreacted with BCN-FITC selectively,whereas tRNAIle2 BCN-Cy5 did not label any RNAinthe 293T transcriptome without AGNmodification did not react (Figure 4Aand 4B). (Figure 5C and 5D). When cells expressed tRNAIle2-5S,

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Figure 6. Fluorescent imaging of Af tRNAIle2-5S in U2OS cells. Upper panel:Cy5 fluorescence (left), DAPI fluorescence (nuclear staining; middle), and differential interference contrast (DIC) imaging (right) of U2OS cells harboring the pCMV-Myc-Tias plasmid. Lower panel:Cy5 fluorescence (left), DAPI fluorescence (nuclearstaining;middle), and DIC imaging (right) of U2OS cells harboring the pCMV-Myc-5S-Tias plasmid. DAPI= 4’,6-diamidino-2-phenylindole.

the RNAmodifying enzyme Tias to enable the highly specific conjugation of biophysical probes to any RNAofinterest in mammalian cells.This new technique should be useful for investigating RNAfolding, RNA/protein interactions,RNA Ile2 Figure 5. A) Sequence and secondary structure of tRNA in the transportation, and RNAmodification, as well as for con- presence of 3’ and 5’ extensions(denoted tRNAIle2-3-5). B) AGN- Ile2 structing RNA-based nanomaterials,invitro and in mamma- modified tRNA -3-5 is visualized through strain-promoted click reac- [1] tion with BCN-FITC. The bottom panel (EB staining) shows that lian cells. equivalent amounts of tRNAIle2-3-5 with and without AGN modification In comparison to noncovalent RNAimaging methods [2] were loaded. C) Structure of BCN-Cy5. D) Tias specifically modify such as use of the Spinach aptamer, the major advantages of tRNAIle2-5S with AGN in the 293T cells transcriptome, which allows the Tias strategy are its specificity,highly affinity,and subsequent strain-promoted click reaction with BCN-Cy5. The bottom versatility.The Tias labeling strategy relies on the selective panel (EB staining) shows that equivalent amounts of total RNA with recognition of Af tRNAIle2 by Tias (which are both absent in and without tRNAIle2-5S expression were loaded. mammalian cells). Since acovalent approach is then employed after an azide or alkyne group is introduced, however, BCN-Cy5 specifically labeled tRNAIle2-5S.Reverse acovalent bond is created between the fluorophore and target transcription polymerase chain reaction (RTPCR) experi- RNA. This would potentially allow the imaging of low- ments confirmed that tRNAIle2-5S existed as afull-length abundance RNA. By contrast, the noncovalent interaction fusion RNAinthe 293T cells (Figure S15). To further between the GFP chromophore analogue and the Spinach demonstrate the utility of our method for RNAimaging in aptamer has affinity only in the micromolar range.[2] Finally, mammalian cells,U2OS cells expressing Tias with or without the Tias strategy enables versatile site-specific RNAlabeling tRNAIle2-5S fusion RNAexpression were grown in the with fluorescent/IR/NMR/EPR agent through click chemis- presence of 2-propynylamine and then fixed, permeabilized, try,without changing the Tias protein or tRNAIle2 sequence. and incubated with Sulfo-Cy5-azide and aCuIcatalyst. We Thechallenge,however, is to develop fluorogenic cyclooctyne found that only the cells expressing tRNAIle2-5S fusion RNA dyes that are membrane permeable,reasonably hydrophobic, could be labeled and imaged with Sulfo-Cy5-azide (Figure 6). and suitable for live cell RNAimaging.Weare currently These results demonstrate that in mammalian cells,Tias can working to achieve this goal. recognize the unique Af tRNAIle2 sequence in the tran- scriptome,thus enabling the sequence- and site-specific conjugation of an azide/alkyne group to an RNAofinterest Ile2 I Experimental Section bearing the tRNA tag.Strain-promoted or Cu -catalyzed In vitro modification of Af tRNAIle2 :ModificationofAftRNAIle2 was click chemistry then enables the specific labeling and imaging performed with Tias (8 mm), Af tRNAIle2 (1 mm), Tris-HCl (100 mm),

of the RNAofinterest in the cells. pH 8.0, KCl (10 mm), MgCl2 (5 mm), DTT (5 mm), ATP(1mm), and Agreat deal of modern RNAbiology and RNAbiotech- substrates 1, 2, 3,or4(1 mm). AGM(1)was used as apositive control. nology involves the site-specific incorporation of modified Each reaction mixture was incubated at 378Cfor 2h.A5mLaliquot nucleotides into RNAmolecules through chemical from each reaction mixture was analyzedby6.5 %acid–urea PAGE (0.1m sodium acetate,pH5.0, 8m urea). In-gel fluorescence images approaches.[1] However,the length of synthetic RNAis were taken at aKFBIO-2800 gel imager and the gel was then stained typically less than 30–50 nucleotides owing to limitations in with ethidium bromide (EB). coupling efficiency. Our new method relies on the RNA Specific fluorescent labeling of Af tRNAIle2 in vitro:The AGN transcription machinery and the high sequence specificity of (2)-modified Af tRNAIle2 (1 mm)was mixed with BCN-FITC (100 mm)

4600 www.angewandte.org 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Angew.Chem.Int. Ed. 2015, 54,4597 –4602 Angewandte Chemie in the buffer (20 mm Tris,pH7.4, 150 mm NaCl) and incubated for Natori, M. Sato,Y.Umezawa, Nat. Methods 2007, 4,413 –419; 30 min at room temperature.The 2-propynylamine (3)-modified Af h) K. Y. Han, B. J. Leslie,J.Fei, J. Zhang,T.Ha, J. Am. Chem. tRNAIle2(1 mm)was mixed with Sulfo-Cy5 azide (Lumiprobe,B3330) Soc. 2013, 135,19033 –19038;i)K.Sharma, J. J. Plant, A. E.

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