DOI: 10.1002/cbic.201000466

Click Chemistry for Rapid Labeling and Ligation of RNAEduardo Paredes and Subha R. Das*[a]

Dedicated to Professor Stewart W. Schneller on the occasion of his 60th birthday.

Introduction

Among several reactions that conform to the tenets of clickchemistry—those having wide scope and high yield and thatrapidly furnish a single product under ambient conditions—the term is epitomized by the copper(I)-catalyzed azide–alkynecycloaddition (CuAAC) reaction.[1–4] Reports by the Sharplessand Meldal groups that the azide–alkyne 1,3-dipolar cycloaddi-tion can be rapidly catalyzed by CuI,[2, 3] the use of ascorbate asa mild reductant that provides a practical alternative to oxygenfree conditions with CuII salts,[3] and various ligands, particularlytris-(benzyltriazolylmethyl)amine (TBTA) that significantly accel-erate the reaction and stabilize the CuI [5] have led to the wide-spread use of the CuAAC reaction.[4, 6, 7] This click chemistrywith rapid reaction of the azide and alkyne groups to form acovalent triazole linkage without cross reacting with otherfunctional groups has been used to great effect in bioconju-gation chemistry, and an extensive body of literature exists forsuch click chemistry with proteins, glycans, and DNA.[4, 8–12]

However, RNA has an inherently much more labile ribophos-phate backbone due the 2’-hydroxyl vicinal to the internucleo-tide phosphate, and this has likely deterred researchers fromusing CuI click chemistry on RNA. Indeed, the first report ofclick chemistry on RNA (with free 2’-OH) is Jao and Salic’s invivo labeling of transcripts that randomly incorporate 5-ethy-nyluridine.[13] In their report, treatment of fixed cells with CuI

and azide dye permitted the visualization of cellular transcriptsthough following the labeling reaction; the RNA was not re-quired to be intact. More recently, while our report was underreview, Brown and co-workers, who have extensively studiedclick chemistry with DNA,[10] extended the method to RNA. El-Sagheer and Brown report the use of click chemistry to createDNA–RNA hybrids crosslinked through nucleobases and alsobackbone ligation of two strands to generate a functionalRNA.[14]

In our concurrent efforts, we aspired to harness the powerof the click-chemistry approach to bioconjugations with RNA

(with free 2’-OH) while keeping the RNA intact. At the outsetwe sought to label RNA with commercially available Click-ITdyes that include azido or alkynyl groups. Current methods tolabel RNA, either with fluorophores, other probes, or evenazido groups, typically are restricted to synthetic oligonucleo-tides that include a thiol or amino modification in the se-quence that is treated with a maleimide or N-hydroxysuccini-mide (NHS) esters.[15, 16] The latter is more common and lesscomplicated, nevertheless the reactions typically take up tofour hours, cannot be used with amino groups in the reaction(thereby precluding TRIS buffer), and require slightly alkalinepH that is optimal for NHS ester stability and reactivity.[16] Theability to use click chemistry directly to label RNA would offera more rapid process, an important consideration for handlingRNA, as well as providing an orthogonal method to the currentNHS chemistry that could enable more facile dual labeling ofoligonucleotides. Thus the rapid and selective click chemistrymethod would represent a significant addition to the reper-toire of RNA functionalization tools and enhance the biologicaldetection, study and delivery of RNA.

Results and Discussion

Acetonitrile cosolvent promotes click chemistry viable withDNA and RNA

Initially—in the absence then of any report of intact RNAunder click chemistry conditions—we tested the stability of

The copper(I)-promoted azide–alkyne cycloaddition reaction(click chemistry) is shown to be compatible with RNA (withfree 2’-hydroxyl groups) in spite of the intrinsic lability of RNA.RNA degradation is minimized through stabilization of the CuI

in aqueous buffer with acetonitrile as cosolvent and no otherligand; this suggests the general possibility of “ligandless” clickchemistry. With the viability of click chemistry validated on syn-thetic RNA bearing “click”-reactive alkynes, the scope of the re-

action is extended to in-vitro-transcribed or, indeed, any RNA,as a click-reactive azide is incorporated enzymatically. Onceclickable groups are installed on RNA, they can be rapidly clicklabeled or conjugated together in click ligations, which may beeither templated or nontemplated. In click ligations the resul-tant unnatural triazole-linked RNA backbone is not detrimentalto RNA function, thus suggesting a broad applicability of clickchemistry in RNA biological studies.

[a] E. Paredes, Prof. S. R. DasChemistry Department, Carnegie Mellon University4400 Fifth Avenue Pittsburgh, PA 15213 (USA)Fax: (+ 1) 412-268-1061E-mail : srdas@andrew.cmu.edu

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/cbic.201000466.

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RNA under reported conditions that are typical for click reac-tions with DNA,[10, 17, 18] with a view to extending this approachto RNA. We observed that both DNA and RNA remain intactover 5 h in buffered solutions (100 mm Tris·HCl; pH 7.5) whenincubated with a large excess of CuI generated by in situ re-duction of copper sulfate with sodium ascorbate—but only ifthe solutions used are first degassed to remove dissolvedoxygen and reduce the risk of oxidative degradation of the oli-gonucleotides (Figure S1 in the Supporting Information). Wethen tested these conditions on click reactions with synthe-sized DNA and RNA that included a 5’-terminal hexyne with atwofold excess of a fluorescent dye azide (Figure 1 A). The en-suing azide–alkyne cycloaddition is slower than the rapid deg-radation of DNA and RNA (data not shown), likely due to CuI

disproportionation and accompanying aqueous redox chemis-try.[19] This is consistent with reports on click chemistry withDNA;[17, 18, 20] furthermore, solutions buffered at lower pH afford-ed no obvious decrease in the likely hydroxy radical-mediatedRNA degradation (data not shown). To minimize degradation,we turned to ligands that are known both to stabilize CuI andaccelerate the reaction. As we were using excess CuI, the reac-tion acceleration was less of a concern than the ability of theligand to stabilize CuI and maintain DNA and RNA integrity.The most widely used ligand TBTA[5] has poor water solubility,and its water-soluble analogue tris-(3-hydroxypropylltriazolyl-methyl)amine (THPTA),[21]—used by Brown and co-workers forDNA[10] and more recently RNA[14]—is not commercially avail-able. We therefore focused our attention on a few other CuI-stabilizing ligands that are readily available, have good water

solubility and have been shown to promote the click reac-tion.[21, 22] These ligands (see Figure 1 B) were used in concentra-tions equimolar to the CuI. In addition, as click chemistry is re-ported in acetonitrile (ACN) as the solvent, which is known tocoordinate and stabilize CuI,[6, 23, 24] we tested the click reactionin the presence of ACN as cosolvent and without any otherligand. We found the use of 2 % acetonitrile in buffer to be aseffective as any ligand in the click labeling of 5’-hexynyl-DNAwith azido AlexaFluor 594 dye without any apparent degrada-tion within 5 h (Figure 1 B).

However, after the five hours of reaction required for DNAclick labeling, little RNA is left intact (data not shown). Multiplefluorescent bands visualized on polyacrylamide gels suggestedthat although RNA was being labeled, it was being degradedas well. To reduce the reaction time and facilitate the cycload-dition reaction over RNA degradation, we increased the con-centration of the reacting species to 1 mm hexynyl-RNA and3 mm azide AlexaFluor 594, included a much larger excess ofCuI over the RNA (�15 equivalents) and increased the propor-tion of CuI-stabilizing acetonitrile cosolvent. The use of15 equivalents of CuI in a 30 min reaction, followed by dena-turing PAGE of the reaction mixture, afforded a single fluores-cent band. The fluorescent band was excised and eluted into abuffered solution, and the absorbance spectrum showed a oneto one correspondence of peaks for RNA and AlexaFluor 594(Figure 1 C), thus suggesting that the RNA was click labeled. Atconcentrations of RNA in the low micromolar range, we couldclick label RNA in an hour with negligible degradation byusing CuI in millimolar concentrations and 15 % acetonitrile or

Figure 1. Click chemistry with DNA and RNA. A) Scheme to test click labeling of a 5’-hexynyl DNA or RNA to an azide dye (see text and the Supporting Infor-mation for specific reaction conditions and Table 1 for sequences). B) Dried 10 % polyacrylamide gel (8 m urea) under UV illumination (365 nm) shows success-fully click-labeled 22-mer 5’-hexynyl DNA2 with AlexaFluor594-azide (lane 0) that appears as a single fluorescent spot between xylene cyanol (XC) and bromo-phenol blue (BFB) markers; lanes 1–3: with CuI-stabilizing ligands 1–3, respectively, or lane 4: 2 % acetonitrile cosolvent without ligand in the reaction. Thestructures of TBTA and its water-soluble analogue THPTA, as well as PMDETA (1), 4,4’-dimethyl-2,2’-dipyridyl (2), bathophenanthroline disulfonic acid (3), andacetonitrile (4) are shown on the right. C) Absorbance spectrum of nonamer 5’-hexynyl RNA2 and AlexaFluor 594-azide after the click reaction, gel electro-phoresis, and elution of the excised band. After click labeling, quantitation of the nonamer RNA (e260 = 93 500 m

�1 cm�1) at 260 nm and AlexaFluor at 590 nm(e590 = 90 000 m

�1 cm�1) shows a 1:1 correspondence. D) 10 % polyacrylamide gel (8 m urea) sandwiched between plastic wraps shows successful, optimizedclick labeling of the nonamer RNA2 with Cy5-azide, the product appears as a single fluorescent band next to the BFB reference lane when using CuI-stabiliz-ing PMDETA (lane 1) or 20 % acetonitrile cosolvent without ligand (lane 4) in the reaction and loaded without any other dye. The RNA2-Cy5 band can be ex-cised and eluted for further use.

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ligand equimolar to CuI. In all our subsequent click reactions,we used either 20 % acetonitrile (for ease in calculating anddispensing volumes for reactions) as a cosolvent or equimolarN,N,N’,N’,N’’-pentamethyldiethylenetriamine (PMDETA) ligandto stabilize the CuI. Additionally, following the success of ourlabeling gels for analysis of the RNA labeling method, as wesought to label RNA fluorescently for subsequent use, weturned to the use of azide–cyanine dyes. In these RNA labelingreactions, the mixture can be loaded directly on a polyacryl-amide gel to remove the CuI—only the dye-labeled RNA butnot the unreacted dye migrates into the gel (Figure 1 D andFigure S2). The RNA band can then be excised and eluted forfurther use, as in typical RNA gel purification. In a more rapidmethod, the reaction mixture is simply passed through a C18Sep-Pak cartridge, commonly used to desalt RNA, to removethe CuI as well as any unreacted dye that remains in the car-tridge.

Enzymatic incorporation of “clickable” azido groups ontoRNA 5’ and 3’ termini

RNA can be synthesized with terminal 5’-, terminal 3’- or inter-nal 2’-O-alkyne (Figure 2 A) by using commercially available re-agents. Azides are unstable under solid-phase synthesis condi-tions, so azido groups can be introduced into synthetic RNAonly at the 5’ terminus by the method of Miller and Kool forDNA[14, 25, 26] or by using amino-modified sequences that arepostsynthetically derivatized with NHS ethers of azidoalkyl

acids. We therefore sought enzymatic methods to incorporateazido groups into RNA. This would further expand the applica-bility of the click-chemistry approach beyond use with synthet-ic RNA (as described above and by El-Sagheer and Brown)[14] toany natural RNA. Direct-labeling methods for RNA use modifiedtranscriptional initiators that require complex synthesis foreach labeling molecule and are not widely used.[16]

Even so, RNA can be obtained by typical in vitro T7-RNApolymerase transcriptions primed with GMP or other guano-sine analogues as initiators to replace GTP at the 5’ ter-minus.[27–32] We therefore synthesized 5’-azido-5’-deoxyguano-sine (5’-N3G) in two steps from guanosine by adapting knownmethods[33, 34] (Scheme S1) and included 5’-N3G in fourfoldexcess over GTP to prime transcriptions (Figure 2B i). The tran-script with the 5’-terminal azide could subsequently be used inclick reactions.

Following our efforts to enzymatically incorporate a 5’-termi-nal azide, we sought to install a click-compatible tag on the3’ terminus of RNA. Radiolabeling of the 3’-end of RNA is typi-cally done with cordycepin (3’-deoxyadenosine) [a-32P] triphos-phate with poly(A) polymerase.[35] We therefore tested thestandard poly(A) polymerase RNA 3’-end-labeling protocol byusing commercial 3’-azido-2’,3’-dideoxytriphosphate (3’-N3dATP; Figure 2B ii). The incorporation of a 3’-terminal azideallows for subsequent click reactions and the use of poly(A)polymerase greatly expands the scope for click-chemistry con-jugations with RNA as it can be performed on any RNA—whether synthetic, in vitro transcribed, or obtained from cellu-

lar extract.Any RNA (with free 2’-hydroxyl groups), synthetic

or otherwise, can thus be readily labeled or conjugat-ed with a wide variety of commercially available dyesor other molecules such as lipids or biotin. Click la-beling should be generally useful for a variety of bio-conjugation applications. For example, postsyntheticRNA can be click labeled to cyanine dye and rapidlydesalted without gel purification. Click-labeled RNA-dye loaded and run on a typical polyacrylamide gelcan be imaged directly (i.e. , without having to drythe gel and expose it to a phosphorscreen). With aCy5-labeled RNA in a polyacrylamide gel, we coulddetect as little as 500 fmol of RNA on a Fujifilm FL-5100 imager (Figure 2 C and Figure S3). In addition,as the conditions for click labeling are orthogonal tothe NHS chemistry that is currently used for the post-synthetic labeling of amino-modified RNA, thesemethods may be combined for dual site-specificfunctionalization of RNA.

Nontemplated click ligation of RNA

The viability of click chemistry with RNA and accessto both RNA with an alkyne (synthetic) and RNA withan azide (enzymatic) suggested the feasibility ofRNA–RNA conjugations (Figure 3 A). A crude RNAtranscript with a putative 5’-azide (13 mm) was sub-jected to a click reaction with a synthetic RNA bear-

Figure 2. Click labeling of RNA A) using synthetic RNA functionalized with commerciallyavailable reagents to include i) terminal 5’-hexynyl, ii) terminal 3’-propargyl, or iii) internal2’-propargyl groups for post-synthetic click chemistry or B) with enzymatically incorporat-ed azide groups on RNA termini i) by transcription with T7 RNA polymerases that include5’-N3G to furnish 5’-azido RNA and ii) by poly(A) polymerase 3’-end tagging reactionswith 3’-N3ddATP to provide RNA with 3’-terminal azide. C) Detection of Cy5-labeled RNAwith gel scanned on a Fujifilm FLA-5100 instrument directly after electrophoresis. RNAruns below xylene cyanol (XC) and was included in the following amounts in lanes 1–6:50 fmol, 250 fmol, 500 fmol, 1 pmol, 2.5 pmol, 5 pmol, respectively. The scan was takenat 800 V detector voltage.

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ing a 3’-terminal alkyne (50 mm). This click ligation of the twoRNAs included 5 mm CuI and 20 % acetonitrile as cosolvent—15 % or more ACN disrupts base pairing and hybridization(data not shown). The click ligation of the two RNAs is thusnontemplated and thereby similar to the click labeling. Theclick ligation proceeds in 1.5 h with complete conversion ofthe transcript to a larger fragment observed on a polyacryl-amide gel with very little degradation (Figure 3 B). The smallamount of unreacted transcript in the click ligation suggeststhat 5’-N3G completely replaces GTP as the transcription initia-tor and thus the ligation is highly efficient.

Such rapid RNA click ligation with acetonitrile is striking as“splint” ligations, in which the RNA strands are annealed to acomplementary DNA strand that spans both RNAs and ligatedwith DNA ligase, require significant optimization of the anneal-ing and ligation conditions and comparatively long hybridiza-tion and ligation times, particularly when the RNAs being ligat-ed are highly structured.[36, 37] The use of acetonitrile to stabilizeCuI is thus doubly advantageous as, in addition to stabilizingCuI, it disrupts any potentially impeding RNA secondary-struc-ture formation, and click chemistry can furnish ligated RNAswith no additional optimization in less than two hours.

Templated RNA click ligation

To investigate whether the use of a splint accelerated the clickligation reaction[38] and to test if the unnatural triazole linkagedeters proper RNA folding, we carried out a templated click li-gation of two fragments of the biochemically well describedtrans-acting hepatitis delta virus (HDV) ribozyme.[39, 40] Poly(A)polymerase was used to introduce a 3’-terminal azide onto anRNA transcript corresponding to a 5’-segment of the ribozyme.This segment RNA of the HDV ribozyme has no cleavage activi-ty as the (untranscribed) 3’-remainder of the HDV sequencecontains the cytosine residue (C76) that is critical for catalyticactivity.[40, 41] This 3’-fragment RNA containing C76 was synthe-sized with a 5’-terminal hexyne (Figure 4 A). As the two RNAsare part of the HDV ribozyme that forms a base-paired stem-loop near the click ligation site, we annealed the two frag-ments and performed the click ligation reaction using PMDETA

as the stabilizing ligand ratherthan acetonitrile to allow thesecondary structure to self-tem-plate the click ligation.[38, 42] Inthe ligation with 3 mm 5’-sectionRNA (with a putatively incorpo-rated 3’-azide) and 13 mm 5’-hex-ynyl-3’-fragment RNA, we couldobtain in one hour, a click-ligat-ed RNA of length similar to thatof the fully transcribed trans-acting HDV (WT) ribozyme (Fig-ure 4 B). Following the click liga-tion reaction, the RNA wasloaded onto a polyacrylamidegel, and the band correspondingto the transcribed WT ribozyme

was excised and eluted. This templated click ligation is fasterthan the one we had observed in the nontemplated click liga-tion and with less degradation.

As the click ligation yields RNA with an unnatural triazolelinked backbone, we were curious as to its effect on RNA struc-ture and function. A disulfide-linked Varkud satellite ribozyme

Figure 3. Nontemplated RNA ligation A) ligation scheme B) aliquots of the RNAs in the click ligation reaction withethidium stain on a 10 % polyacrylamide gel illuminated with UV (365 nm) indicate that RNA3 from transcription(lane 1) is click ligated (lane 2) to yield a larger RNA. The short synthetic RNA4 does not stain and the loading dyeXC runs below the portion of the gel shown.

Figure 4. Templated click ligation of RNA. A) Secondary structure of a trans-acting ribozyme derived from the HDV obtained by click ligation. The 5’-seg-ment of the ribozyme (black) was transcribed. The adenosine (A) that pro-vides the terminal 3’-azide was incorporated by using poly(A) polymerase.Annealing to a synthetic 5’-hexynyl-3’-fragment RNA (blue) that includes the“catalytic” C76 residue allows the templated click ligation reaction withPMDETA. The inset shows a schematic of the expected triazole-linked back-bone following the click ligation. The ribozyme cleaves an RNA substrate(red) at the indicated site (see Table 1 for sequences). B) Ethidium-staineddenaturing 10 % polyacrylamide gel shows 5’-phosphohexynyl-3’-segmentRNA7 (lane 1), 3’-azido-5’-segment azide RNA6 (lane 2), and an aliquot of theclick ligation crude reaction mixture after 1 h (lane 3). RNA6 is absent inlane 3, and the crude click ligation product runs similarly to the fully tran-scribed purified trans-HDV ribozyme (lane 4). C) The 5’-segment RNA displaysno detectable cleavage activity (*) while the single-turnover cleavage kinet-ics (pH 7.5, 10 mm Mg2+ , 25 8C) of the click-ligated ribozyme (*) are nearlyindistinguishable from those of the full-length transcribed HDV ribozyme(^). A two-piece HDV ribozyme requires a twofold excess of the catalyticC76 strand and required separately optimized conditions to obtain cleavagekinetics (*) that were slower than the WT or click-ligated ribozyme.

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was shown to be catalytically competent with judicious choiceof the unnatural linkage.[43] The click ligation site in our HDVribozyme was chosen at a stem-loop that is known to be non-essential for HDV function.[39] Still, in a trans-acting ribozymeconstructs in which the catalytic C76 is on a separate strand,excess of the catalytic strand and optimization of folding andannealing conditions are required.[44] Therefore, it was unclearwhether the click-ligated HDV RNA, which is a single RNAstrand, though with an unnatural backbone triazole, wouldfold adequately and be catalytically competent. We thereforetested the click-ligated HDV ribozyme for RNA cleavage activitywith 10 mm Mg2+ under the same standard conditions report-ed for the WT ribozyme without optimization. The reactionrate and extent of cleavage of the RNA substrate by the click-li-gated HDV ribozyme (0.84�0.09 min�1; 62 %) are nearly indis-tinguishable from those of the transcribed WT HDV ribozyme(0.88�0.08 min�1; 59 %; Figure 4 C). This suggests that, with asuitably chosen ligation site, the unnatural triazole-linked back-bone does not deter competent folding and function of RNA.

El-Sagheer and Brown used a similar backbone-ligation strat-egy to generate a DNA–RNA hybrid hammerhead ribozyme ina powerful example of the biocompatibility of the backbonetriazole linkage.[14] They showed that a ribozyme that includesa deoxyribose modification and the triazole linkage close tothe active site is still catalytically competent. Thus, althoughnon-native, click-ligated RNA can provide near-native RNAfunction. A two-piece (unligated) HDV ribozyme requires an-nealing with excess of the catalytic C76 strand and suitably op-timized conditions[44] and is still moderately less active (0.32�0.1 min�1; 44 %) than the WT or click-ligated ribozyme (Fig-ure 4 C). Loops regions such as the one we chose for the clickligation site are common and used in both small and largefunctional RNAs as sites for modification.[41, 45–47] Our resultssuggest that with such a suitably chosen ligation site, click-li-gated RNA can be obtained rapidly and handled without otheroptimization, in a similar fashion to transcribed RNA.

Conclusions

We have shown that click chemistry may be used on RNA withfree 2’-hydroxyl groups in spite of the need for CuI. Degrada-tion of the RNA can be prevented by degassing and the use ofeither a CuI-stabilizing ligand or simply acetonitrile as cosol-vent. The use of acetonitrile as cosolvent in low amounts (2 %was sufficient in for DNA) raises the possibility of using theseconditions for “ligandless” click chemistry in general. Clickablegroups can be installed on RNA either through synthesis or byenzymatic incorporation at the termini. Once the clickablegroups are installed, rapid click labeling or click ligations oftwo RNAs is possible in nontemplated or templated reactionsas described here and recently by El-Sagheer and Brown.[14]

The ability to ligate RNA rapidly to incorporate synthetic modi-fications could be useful in RNA functional and structural biol-ogy for the site-specific incorporation of modified residues andatoms.[48, 49] As all RNA and molecules used in this report canbe obtained from commercial sources (including 5’-N3G), weenvision this report on click labeling and ligation can be readi-

ly used to label and conjugate molecules rapidly to syntheticas well as natural RNA for biological studies.[50] The enzymaticincorporation of click-chemistry-compatible tags into RNA—synthetic, in-vitro-transcribed or cellular in origin—allows forclick labeling or conjugation to a host of molecules for the de-tection, manipulation, analysis, or delivery of the RNA. Triazole-linked lipid–oligonucleotide conjugates have been shown tobe nontoxic;[51] this suggests a broad applicability of clickchemistry to any synthetic or natural RNA. Coupled with newdevelopments in copper-free click labels[52, 53] that will make invivo conjugations even more facile, the method for installingclick-compatible tags onto RNA described here might empow-er new efforts in RNA chemistry and biology.

Experimental Section

Chemicals and general experimental: Commercially availablecompounds were used without further purification. Phosphorami-dites with labile labile phenoxyacetyl (PAC) protecting groups andappropriate reagents for the solid-phase synthesis of DNA andRNA were purchased from ChemGenes (Wilmington, MA USA) orGlen Research (Sterling, VA USA). RNA- and DNA-synthesis columnswere purchased from Biosearch Technologies, Inc. (Novato, CAUSA). The 5’-hexynyl modifier phosphoramidite was purchasedfrom Glen Research. 3’-O-propargyl CPG columns and guanosinewere purchased from ChemGenes. Triphenylphosphine and I2 werepurchased from Sigma–Aldrich. Dichloromethane was purchasedfrom Fisher. Imidazole was purchased from Ameresco (Framing-ham, MA USA). Sodium azide, N-methyl-2-pyrrolidone (NMP), anddimethylformamide (DMF) were purchased from Alpha Aesar. Nu-cleotide triphosphates (NTPs) were purchased from Fermentas, and3’-azido-2’,3’-dideoxyadenosine triphosphate (3’-N3ddATP) was pur-chased from Trilink BioTechnologies (San Diego, CA USA). T7 RNApolymerase and Poly(A) polymerase were purchased from NewEngland Biolabs. Copper sulfate pentahydrate (CuSO4·5 H2O) andCuI-stabilizing ligands PMDETA, bathophenanthroline disulfonicacid (BPDSA), and 4,4’-dimethyl-2,2’-dipyridyl (DMDP) were pur-chased from Sigma–Aldrich. HPLC-grade acetonitrile was pur-chased from Fisher. Sodium ascorbate was purchased from AlfaAesar. All other solvents and reagents were purchased from Fisher.Azide AlexaFluor 594 was purchased from Invitrogen. Azide cya-nine 5 (Cy5) and azide cyanine 3 (Cy3) were purchased from Lu-miprobe (Hallandale Beach, FL, USA). DNA and RNA were obtainedfrom Integrated DNA Technologies (Coralville, IA, USA), Dharmacon(Fisher) or synthesized (see below).

Infrared spectra were obtained on a JASCO FTIR 6300 instrument,while 1H NMR spectra were recorded on a Bruker Avance 300 MHz

instrument and analyzed by using Topspin 1.2 software.

Synthesis of 5’-azido-5’-deoxyguanosine: 5’-N3G was synthesizedfrom guanosine in two steps by adapting literature procedures(Scheme S1).[12, 13] I2 (14 g 55 mmol) was added in portions over10 min to a solution of guanosine (5 g,16.6 mmol), Ph3P (14.4 g,55 mmol) and imidazole (7.5 g, 110 mmol) in NMP (66 mL). After4 h of stirring, the product was precipitated by the addition ofCH2Cl2 (600 mL) and water (200 mL), and the solution was filteredto obtain 3.95 g (56 %) of 5’-deoxy-5’-iodoguanosine as a yellowsolid. The TLC and 1H NMR (data not shown) matched reportedvalues.[12]

NaN3 (1 g, 38 mmol) was added to 5’-deoxy-5’-iodoguanosine (2 g,19 mmol) in anhydrous DMF (20 mL), and the mixture was stirred

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at 80 8C for 20 h under argon. Following solvent evaporation andthe addition of water (30 mL), the resulting solid was collected byvacuum filtration and washed with water (30 mL), cold ethanol(9 mL) and cold diethyl ether (6 mL) to give 780 mg (50 %) of 5’-N3G, which was characterized by IR and 1H NMR that match report-ed values[13] (spectra 1 and 2 in the Supporting Information).

Nucleic acid synthesis and preparation: Solid-phase oligonucleo-tide synthesis was performed on a MerMade–4 instrument (Bio-automation, Plano, TX, USA). Synthesis and deprotection of the oli-gonucleotides was conducted under standard protocols for PAC-protected amidites, as recommended by the manufacturer. In vitrotranscriptions were conducted with T7 RNA polymerase fromsingle-stranded DNA templates with appropriate promoter sequen-ces by using standard procedures.[31] In some cases, transcriptionswith 5’-N3G were primed with 5’-N3G in 4:1 excess over GTP.

Where necessary, 5’-end radiolabeling was conducted with T4 PNKby using standard protocols. Azide tagging of the 3’-end was per-formed with poly A polymerase by using 3’-N3ddATP in place ofcordycepin (3’-deoxyadenosine) [a-32P]triphosphate as used instandard 3’-end labeling protocols.[16] RNAs were purified by PAGEwith 8 m urea. The RNA bands were excised and eluted overnightin buffer (pH 7.5, 10 mm Tris/0.1 mm EDTA) at 4 8C, and precipitatedin ethanol. The DNA and RNA sequences used in this study aresummarized in Table 1.

Degassing of solutions: Stock solutions of Cu2SO4, sodium ascor-bate, and Tris·HCl buffer (pH 7.5) were degassed by bubblingargon through the solutions for 15 min prior to adding DNA orRNA. Trace amounts (<50 nmol) of 5’-radiolabeled DNA1 andRNA1 were incubated in buffered CuSO4/ascorbate solution withand without degassing. Little breakdown of input DNA or RNA wasobserved in the degassed solutions (Figure S1).

DNA and RNA click labeling and detection: Click labeling ofDNA2 (10 mm) was conducted by using Tris·HCl (100 mm, pH 7.5),azide AlexaFluor 594 dye (20 mm), CuSO4 (20 mm), sodium ascorbate(40 mm), and one of the following: 1) PMDETA (20 mm), 2) BPDSA(20 mm), 3) DMDP (20 mm), or 4) ACN (2 %). The reaction was al-lowed to run for 5 h in degassed solutions and blanketed byargon. The reaction mixture was loaded directly on a 10 % poly-acrylamide gel (8 m urea) to both stop the reaction and resolve theDNA. The gel was dried and illuminated with a hand-held, 4 W,

365 nm UV lamp (UVG-11) to visualize and determine DNA clicklabeling (Figure 1 B).

Several conditions for click labeling RNA2 (10 mm) were tested withACN as the cosolvent and the protocol for DNA click labeling de-scribed above. With 2 % ACN and no fluorescently labeled RNA,complete degradation of the RNA was observed (data not shown);with 10 % ACN, multiple fluorescent bands suggested that some la-beling had occurred; however, the RNA was extensively degraded(data not shown).

RNA click labeling could be observed without RNA degradation byincreasing the concentrations of reactants to RNA2 (1 mm), azidedye (3 mm, AlexaFluor 594, Cy3 or Cy5), sodium ascorbate (15 mm),and CuSO4 (15 mm). The reactions included ACN (15 or 20 %) as co-solvent or PMDETA (15 mm) and were carried out for 30 min withshaking. The reaction mixture was loaded on a urea 10 % polyacryl-amide gel (8 m urea) to resolve and purify the RNA or, in the caseof labeling with Cy dyes, was loaded with C18 Sep-Pak cartridge(Waters)—that is commonly used to desalt RNA—and the labeledRNA was eluted with 50 % acetonitrile in water.

Cy5 click-labeled RNA2 was loaded onto a 10 % polyacrylamide gel(8 m urea). Following electrophoresis, the gel was placed betweenplastic wrap and scanned (Figure S3). Detection of 500 fmol perband per lane of Cy5-labeled RNA shows a good signal-to-noiseratio and minimal streaking. More sensitive detection (to 250 fmolRNA) is possible by increasing the detector voltage on the scanner,though the increased background noise might affect quantitation,as lanes with larger amounts exhibit streaking.

Nontemplated click ligation: A click ligation reaction was per-formed with RNA3 (3 mm) in Tris·HCl (100 mm, pH 7.5), RNA4(13 mm), sodium ascorbate (5 mm), and ACN (20 %). The reactionwas initiated by the addition of CuSO4 (5 mm) and allowed to runfor 1.5 h under argon at room temperature. The reaction mixturewas loaded onto a 10 % polyacrylamide gel (8 m urea) to stop thereaction and to resolve and visualize the RNAs (Figure 3 B).

Templated click ligation: Click ligation reactions were performedby using RNA6 (3 mm), RNA7 (13 mm, blue strand in Figure 4 A),CuSO4 (5 mm), and PMDETA (5 mm ; to allow a template assistedreaction). The RNAs were annealed by holding the reaction mixtureat 70 8C for 2 min and then cooling it to room temperature. Thehybridization of the two RNA strands was verified by running an

Table 1. DNA and RNA sequences used in this study. Lower case and upper case letters denote DNA and RNA residues respectively. RNA6 and RNA7 arethe sequences of the HDV ribozyme, and RNA8 is the substrate in Figure 2 B. In RNA7, the underlined C represents the “catalytic” cytosine C76 in the HDVribozyme (see Figure 4 B).

Name Sequence Modifications Source5’- 3’-

DNA1 5’-gtg cca agc tta ccg none none syntheticRNA1 5’-GAU GGC CGG CA none none syntheticDNA2 5’- cga ctc act ata gga aga gat g phosphohexyne none syntheticRNA2 5’-GAA GCG CCA phosphohexyne none syntheticRNA3 5’-GGG CAU CUC CAC CUC CUC GCG GUC CGA CCU GGG azide none in vitro transcription primed with 5’-N3G

CAU CCG AGC ACU CGG AUG GCU AAG GGA GAG CCARNA4 5’-GAA GCG CCA none O-propargyl syntheticRNA5 5’-GGG CAU CUC CAC CUC CUC GCG GUC CGA CCU none none in vitro transcription

GGG CAU CCG AGCRNA6 5’-GGG CAU CUC CAC CUC CUC GCG GUC CGA CCU GGG none azide in vitro transcript (RNA5) end-tagged using

CAU CCG AGC a 3’-N3ddATPRNA7 5’-CUC GGA UGG CUA AGG GAG AGC CA phosphohexyne none syntheticRNA8 5’-UUC GGG UCG GC none none synthetic

130 www.chembiochem.org � 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2011, 12, 125 – 131

S. R. Das et al.

aliquot of the mixture on a native 10 % polyacrylamide gel (datanot shown). The click ligation was started by adding sodium ascor-bate (5 mm), and the reaction allowed to run for 1 h under argon.The RNA was purified by PAGE, as described above, to obtain theclick-ligated HDV ribozyme.

Ribozyme reactions and analyses: The click-ligated HDV ribozymeand the HDV ribozyme (WT) obtained by transcription, as previous-ly reported,[19] were tested in cleavage reactions of substrate RNA8.The cleavage reactions contain ribozyme at saturating concentra-tion (1 mm) to ensure that the observed cleavage rate constantsrepresent single turnover cleavage rates of the enzyme substrate(E·S) complex as described in the literature. Ribozymes (1 mm) insodium 4-morpholinepropanesulfonic acid (MOPS) buffer (50 mm,pH 7.5) containing MgCl2 (10 mm) were preincubated at 70 8C for2 min, then kept at room temperature for 15 min. Reactions wereinitiated by the addition of trace amounts (<1 nmol) of the radio-labeled substrate RNA. Aliquots were removed after 1, 2, 5, 10, and30 min and quenched (formamide/100 mm EDTA stop solution,9:1). The 5’-labeled cleavage product was separated from un-cleaved substrate RNA by 20 % PAGE (8 m urea), the dried gelswere exposed to phosphor screen and imaged on a Storm phos-phoimager (Molecular Dynamics). The cleavage products werequantified and normalized to the sum of the product and substratebands with ImageQuant software. The data for cleavage reactionswere fitted to Equation (1):

y ¼ y0 þ ae�kobs t ð1Þ

where kobs is the observed first-order cleavage rate constant.

Acknowledgements

We thank Catalina Achim for helpful discussion on CuI-stabilizingligands. This work was supported by Carnegie Mellon Depart-ment of Chemistry start-up funds.

Keywords: click chemistry · labeling · ligation · RNA ·transcriptional priming

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Received: August 11, 2010Published online on December 5, 2010

ChemBioChem 2011, 12, 125 – 131 � 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chembiochem.org 131

Click Chemistry for RNA