New strategy for the synthesis of chemicallymodified RNA constructs exemplified byhairpin and hammerhead ribozymesAfaf H. El-Sagheera,b and Tom Browna,1

aSchool of Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ, United Kingdom; and bChemistry Branch, Department of Scienceand Mathematics, Faculty of Petroleum and Mining Engineering, Suez Canal University, Suez 43721, Egypt

Edited by P. H. Seeberger, Max Planck Institute of Colloids and Interfaces, Germany, and accepted by the Editorial Board July 15, 2010 (received for review May9, 2010)

The CuAAC reaction (click chemistry) has been used in conjunctionwith solid-phase synthesis to produce catalytically active hairpinribozymes around 100 nucleotides in length. Cross-strand ligationthrough neighboring nucleobases was successful in covalentlylinking presynthesized RNA strands with high efficiency (trans-ligation). In an alternative strategy, intrastrand click ligation wasemployed to produce a functional hammerhead ribozyme contain-ing a novel nucleic acid backbone mimic at the catalytic site (cis-ligation). The ability to synthesize long RNA strands by a combina-tion of solid-phase synthesis and click ligation is an importantaddition to RNA chemistry. It is compatible with a plethora of site-specific modifications and is applicable to the synthesis of manybiologically important RNA molecules.

chemical ligation ∣ click RNA ligation ∣ CuAAC ∣ triazole backbone ∣biocompatible

Oligonucleotide chemistry is central to the advancement ofcore technologies such as DNA sequencing and genetic

analysis and has impacted greatly on the discipline of molecularbiology (1, 2). Oligonucleotides and their analogues are essentialtools in these areas. They are produced by automated solid-phasephosphoramidite synthesis, a highly efficient process that can beused to assemble DNA strands over 100 bases in length (3).Synthesis of RNA is less efficient owing to problems caused bythe presence of the 2′-hydroxyl group of ribose, which requiresselective protection during oligonucleotide assembly. Thisreduces the coupling efficiency of RNA phosphoramidite mono-mers due to steric hindrance. In addition, side-reactions that oc-cur during the removal (or premature loss) of the 2′-protectinggroups cause phosphodiester backbone cleavage and 3′ to 2′phosphate migration (4). Although several ingenious strategieshave been developed to minimize these problems (5) and to im-prove the synthesis of long RNA (6), the chemical complexity ofsolid-phase RNA synthesis dictates that constructs longer than 50nucleotides in length remain difficult to prepare. Most biologi-cally important RNA molecules such as ribozymes (7), aptamers(8), and riboswitches (9, 10) are significantly longer than this, sonew approaches to the synthesis of RNA analogues are urgentlyrequired. Although RNA synthesis by transcription might seem aviable alternative, it does not permit the site-specific incorpora-tion of multiple modifications at sugars, bases, or phosphates. Incontrast, automated solid-phase RNA synthesis is compatiblewith the introduction of fluorescent tags, isotopic labels (forNMR studies) and other groups to improve biological activityand resistance to enzymatic degradation. The scope and utilityof important RNA constructs can be significantly extended bysuch chemical modifications, and the scale of chemical synthesisis potentially unlimited (11). These are important factors whenplanning structural studies on RNA or the development of ther-apeutic oligoribonucleotides analogues, and should be consid-ered when devising new methods of RNA assembly. Enzymaticligation might appear to be a method of achieving some of these

objectives (12). T4 RNA ligase can be used to join nucleotides insingle-stranded regions such as RNA loops, whereas T4 DNAligase functions on double-stranded RNA. However, neither en-zyme can be used to link together RNA strands at other positions,such as across the bases or sugars, or to create unnatural linkages.The use of ligases has other drawbacks; they are often contami-nated with RNases that can partially degrade the ligation prod-ucts, and the ligation protocols are quite complicated, requiringphenol extraction to remove the protein. Moreover, enzymaticligation methods are not suitable for the large scale synthesis ofRNA, and the yields of enzymaticRNA ligation are often low. Thisis partly due to the tendency of T4 RNA ligase to cyclize phos-phorylated RNA strands and to join together incorrect strands.

Here we describe click RNA ligation, a strategy that overcomesthe above limitations and could lead to a step-change in the synth-esis of biologically active RNA constructs. The CuAAC reaction(13, 14) was chosen for RNA ligation owing to its very high speed,efficiency, orthogonality with functional groups present in nucleicacids and compatibility with aqueous media. In this procedure,individual RNA oligonucleotides are assembled by automatedsolid-phase synthesis, purified by HPLC then chemically ligatedby click chemistry (15) to produce much larger molecules. In thisstudy we demonstrate the power of click ligation by synthesising aseries of chemically modified hairpin and hammerhead ribo-zymes. The hairpin ribozyme belongs to a family of catalyticRNAs that cleave their RNA substrates in a reversible reactionto generate 2′,3′-cyclic phosphate and 5′-hydroxyl termini. It wasdiscovered in tobacco ringspot virus satellite RNA where ribo-zyme-mediated self-cleavage and ligation reactions participatein processing RNA replication intermediates (16). It was chosenfor the current study for three reasons: Its size is such that directchemical synthesis is problematic and therefore click ligation be-comes a valuable technique, its biophysical and catalytic proper-ties have been extensively studied and are well understood (17),and its functionality can be extended by the incorporation of che-mical modifications such as fluorescent tags (18). In this studythree analogues of the hairpin ribozyme were synthesized fromindividual oligonucleotides by crosslinking the side-chains ofmodified uracil bases in opposite strands of the “unclicked” seg-ments (trans-ligation). An RNA ribozyme (98-RNA) was synthe-sized and a DNA/RNA chimera (98-DNA/RNA) was made inwhich segments a and b contain tracts of DNA and segment cis entirely DNA (Fig. 1, Left). Ribozymes are known to tolerate

Author contributions: A.H.E. and T.B. designed research, performed research, analyzeddata, and wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. P.H.S. is a guest editor invited by theEditorial Board.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: tb2@soton.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1006447107/-/DCSupplemental.

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deoxyribonucleosides outside the catalytic site and the incorpora-tion of DNAoffers the possibility of synthesising longer fragmentsfor click ligation. Therefore we were anxious to confirm thatthe CuAAC reaction is compatible with DNA–RNA hybrids. Ashorter 77-nucleotide version of the hairpin ribozyme was alsoprepared (77-RNA, Fig. 1, Right) to evaluate a simple splint-mediated modification of the above click ligation strategy. In adifferent approach, a hammerhead ribozyme (19, 20) was madeby the splint-mediated cis-ligation of two RNA strands (Fig. 2).The hammerhead is a small catalytic RNA motif that is found inplant RNA viruses, satellite RNA, viroids, and repetitive satelliteDNA (21) where it catalyzes cleavage and ligation of RNA duringrolling circle replication (22, 23). It was chosen as a model to in-vestigate the chemistry of a new triazole nucleic acid backbonemimic in click ligation, and to explore its biocompatibility.

Results and DiscussionHairpin Ribozymes. The oligonucleotide building blocks requiredin the assembly of the hairpin ribozyme analogues (segmentsa, b, and c in Fig. 1) were prepared by automated solid-phasephosphoramidite synthesis using 2′-t-butyldimethylsilyl (TBS)protected monomers for the RNA tracts. The TBS method ofRNA synthesis (24) was chosen as it is widely used, the requisitereagents are readily available, and it has been shown to be effec-tive in the synthesis of a wide range of chemically modified RNA

analogues (25). Oligonucleotide sequences are shown in Figs. 1and 2 and in Table S1. For the trans-click ligation reactions, theindividual oligonucleotide segments require an alkyne or azidegroup at the 5-position of a uracil base at the appropriate locus.

Fig. 1. Assembly of hairpin ribozymes by the CuAAC reaction. The substrate cleavage site (þ1 − 1) is indicated by an arrow. 8% polyacrylamide gels: Right-hand lanes show CuAAC crude reaction mixtures and all other lanes show reactants (oligonucleotide segments). The solid arrows point to full-length ribozymeproducts in all three reactions, and the dashed arrow points to DNA splints in the 77-RNA reaction. Gels visualized by UV shadowing. (Left) The 98-mer RNAhairpin ribozyme 98-RNA and DNA–RNA hybrid 98-DNA/RNA. The sequences are the same but 98-DNA/RNA contains deoxyribose sugars at the positionsmarked with asterisks* and has a different pattern of dye-labeling (segment a ¼ 50-cy5, segment b ¼ 50-cy3, segment c ¼ 50-FAM). The ES− mass spectrumof the 98-DNA/RNA ribozyme is shown (found: 33169.4 Da, requires: 33170 Da). (Right) The truncated RNA 77-mer hairpin ribozyme 77-RNA. ES− mass spectrum(found: 26983.6 Da, requires: 26983 Da).

Fig. 2. Structure and sequence of the hammerhead ribozyme (green) andsubstrate (blue) with cleavage site in red. The triazole linkage lies in the cat-alytic pocket opposite to the cleavage site between C3 and U4. NucleotidesC3 and G8 form a base pair in the active conformation of the ribozyme–substrate complex (33).

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The reaction scheme and the structures of the alkyne and azide-labeled nucleoside components 3 and 4 are shown in Fig. 3.Azides are unstable to PIII, and do not survive the conditionsof phosphoramidite oligonucleotide synthesis. Therefore, theazide functionalities were introduced after oligonucleotide as-sembly by selective reaction of the N-hydroxysuccinimide esterof azidohexanoic acid 2 with the primary amino groups of themodified uracil bases (26). All oligonucleotides were purifiedby reversed-phase HPLC and analyzed by mass spectrometryprior to click ligation. For construction of the all-RNA andDNA/RNA 98-mer ribozymes the individual segments were an-nealed and covalently linked in a self-templated CuAAC reaction(27). The CuI catalyst was generated in situ from CuII sulfate andaqueous sodium ascorbate, and a water-soluble CuI-bindingligand 5 was added to prevent RNA degradation (28). Duringthe reaction segment a was crosslinked to segment b near termi-nus B, and segment b to segment c near terminus C across themajor grooves of the ribozyme stems. The crosslinking positionsare indicated in Fig. 1, and the chemical structure of the resultanttriazole linkage 6 is shown in Fig. 3. The two simultaneous clickligation reactions proceeded efficiently in the assembly of bothribozymes, and full-length constructs were purified by polyacry-lamide gel-electrophoresis (Fig. 1, Left). Despite the self-templat-ing, a minor side-reaction occurred due to cyclization of segmentb, which contains both alkyne and azide. This impurity was easilyremoved during ribozyme purification. A shorter 77 nucleotideversion of the hairpin ribozyme was also prepared (77-RNA,Fig. 1, Right), but in this case segment b was synthesized withtwo azide groups, one near the 5′- terminus and the other nearthe 3′-end, rather than alkyne and azide. Segments a and c weresynthesized with an alkyne near their 3′- and 5′-ends respectivelyto ensure chemical complementarity with segment b. This facilechange in strategy eliminated the possibility of unwanted cycliza-tion of segment b and is an example of the flexibility offered bychemical ligation. Segments a, b, and c were labeled with cy5, cy3,and FAM respectively to facilitate fluorescence resonance energytransfer (FRET) studies (7, 29). There are many cases when RNAassembly cannot be self-templated, so an alternative strategy is

necessary. For the smaller hairpin ribozyme 77-RNA we electedto explore the use of two complementary 24-mer DNA splints tohold the short arms of the ribozyme segments in place duringclick ligation. This method gave a clean reaction and the resultantshort hairpin ribozyme was purified by polyacrylamide gel-electrophoresis.

The catalytic activity of the three hairpin ribozyme analogueswas investigated by running cleavage reactions at 55 °C in thepresence of five equivalents of the RNA substrate (Fig. 4). Inall cases five cycles of denaturation (95 °C) followed by cleavage(55 °C) consumed the substrate. It was cleaved at the expectedsite (indicated in Fig. 1), as confirmed by mass spectrometryof the two fragments (ES−, found: 5944 Da, 2154 Da, requires:5943 Da, 2155 Da). The ribozymes also cleaved the substratewithout temperature cycling (Fig. S3). The clicked ribozymeswere robust and could be isolated from the cleavage gels and re-used in subsequent reactions with no significant loss of activity.The fact that the triazole hairpin ribozyme cleaves its substraterapidly and produces the same products as the natural ribozymeis not surprising as the click linkages are located remotely fromthe active site. This design feature can be conveniently incorpo-rated into most large RNA constructs. This click linkage stretchesacross the major groove where it does not perturb the duplex orchange its conformation (26). Cross-strand linking between nu-cleobases in RNA is unique to chemical ligation and cannotbe achieved by enzymatic methods.

The Hammerhead Ribozyme. A minimal hammerhead ribozymewas synthesized by splint-mediated intrastrand click ligation(cis- ligation) of 3′-alkyne and 5′-azide oligoribonucleotides. Thisalternative method was chosen to investigate the suitability ofclick chemistry for linking RNA strands through their backbonerather than between the nucleobases. The success of this strategyin producing an active ribozyme requires the design of a triazolelinkage that closely resembles a phosphodiester group. The struc-ture of this linkage and the synthetic strategy to produce it areshown in Fig. 5. The key building block for the synthesis ofthe 3′-alkyne labeled RNA strand is 5′-DMT-3′-propargyl-5-methyldeoxycytidine 9. This was made in two steps, the firstinvolving 3′-alkylation of 5′-DMT thymidine 7 with propargylbromide in the presence of sodium hydride in THF to give inter-mediate 8 in 82% yield, a considerable improvement on the pub-lished method (30). In the second step, conversion of thymine to5-methyl cytosine was carried out using phosphorus oxychloride,N-methylimidazole and aqueous ammonia. Compound 9 wascoupled to succinylated aminoalkyl solid support to give deriva-tized support 10, which was used in the solid-phase synthesis of3′-propargyl oligonucleotide 11 (Fig. 5). This solid support will beuseful in the synthesis of oligonucleotides for various click liga-tion or labeling applications. The 5′-azide RNA strand 13 wasmade from the corresponding unmodified 5′-hydroxyl RNA con-gener 12 by treatment of the support-bound oligonucleotide withmethyltriphenoxyphosphonium iodide followed by sodium azide.This 5′-azide labeling method has been used previously on DNA

Fig. 3. The synthetic procedure used to covalently link the arms of the hair-pin ribozyme across the major groove of the duplex.

Fig. 4. Hairpin ribozyme substrate cleavage reactions. Lanes 1, 3, 5: cleavagereactions of 98-RNA, 98-DNA/RNA and 77-RNA ribozymes respectively with 5equivalents of substrate. Lanes 2, 4, 6: control—uncleaved substrate. 20%polyacrylamide gels visualized by fluorescence.

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(31) and here we demonstrate its compatibility with RNA. Thetwo components of the hammerhead ribozyme (11 and 13) weredesigned with extra thymidine nucleotides at the termini to aidgel-filtration, whereas the 5′-FAM dye on the DNA splint wasincluded to differentiate it from the clicked ribozyme during re-versed-phase HPLC purification. Splint-mediated ligation of thetwo RNA strands 11 and 13 proceeded smoothly to give ribozyme14 with a triazole linkage in the backbone (Fig. 6A, lane 4). Themodified hammerhead ribozyme was characterized by mass spec-trometry (ES-, found: 7585 Da, requires: 7585 Da) and was shownto cleave its substrate, generating an identical product to thatformed by the normal unmodified ribozyme (Fig. 6B, lanes 1and 2). Cleavage occurred at the predicted scissile phosphodiesterbond between cytidine and adenosine as confirmed by mass spec-trometry of the two fragments (ES-, found: 6490 Da, 1495 Da;requires: 6490 Da, 1496 Da). Parallel experiments on a 5′-fluor-escein labeled substrate demonstrated complete cleavage in30 min at 37 °C, similar to the native ribozyme (Fig. 6C). This isa satisfying result, particularly as the unnatural triazole linkageis located at the active site between residues C3 and U4 of theribozyme (Fig. 2). X-ray structures of the minimal (19, 20, 32)and full-length hammerhead ribozymes (33) confirm the criticalpositioning of C3. It is postulated that a base pair between C3and G8 holds the ribozyme in its catalytically competent confor-mation (33). The observed activity of the triazole ribozyme con-

firms that in this structure the designed linkage is a suitableanalogue of a phosphodiester group.

To further demonstrate that the triazole linkage is a viablesubstitute for a phosphodiester group we carried out UV-melting

Fig. 5. Synthesis of the hammerhead ribozyme. Synthesis of alkyne and azide RNA strands, construction hammerhead, and structure of triazole backbone.

Fig. 6. (A). CuAAC reaction to synthesize the hammerhead ribozyme, 20%polyacrylamide gel: Lanes 1, 2: starting oligonucleotides (alkyne and azide),lane 3: DNA splint, lane 4: crude reactionmixture. (B). Hammerhead ribozymecleavage reaction with nonfluorescent substrate. Lane 1: Sþ TR, lane 2:Sþ NR, lane 3: TR, lane: 4 NR, lane 5: S. (S ¼ uncleaved substrate,NR ¼ normal ribozyme control, TR ¼ triazole ribozyme). Gels A and B visua-lized by UV shadowing. TR (24-mer) is longer than NR (16-mer) due to theadditional thymidines at the termini. (C). Hammerhead ribozyme cleavagereaction with 5′-fluorescein labeled substrate. Lane 1: FS, lane 2: FSþ NR30 min, lane 3: FSþ TR 30 min, lane 4: FS, lane 5: FSþ NR 1 h, lane 6:FSþ TR 1 h. (FS ¼ fluorescent substrate, gel imaged by fluorescence).

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studies on the natural and triazole ribozymes with their RNA sub-strate in the absence of magnesium ions (to prevent substratecleavage). We obtained melting curves that were very similar(Fig. S4), with a slight lowering in melting temperature in thetriazole case (ΔTm ¼ 1.1 °C). We then hybridized the nativeand triazole hammerhead ribozymes to their DNA complementto produce DNA–RNA hybrid duplexes. In this case the triazoleanalogue was almost as stable as its natural counterpart(ΔTm ¼ 3.8 °C) (Fig. S4). The CD spectra of the above constructsindicate that the triazole modification does not change the duplexconformation (Fig. S5). Although it would not generally benecessary to place the triazole modification at a critical catalyticposition when designing RNA constructs we have shown herethat such a substitution can lead to stable biologically activeribozymes, thereby demonstrating the biocompatibility of thetriazole linkage.

ConclusionIn this study click chemistry has been used to ligate presynthe-sized oligonucleotides to construct RNA molecules and DNA/RNA chimeras up to 100 nucleotides in length. The method iscompatible with site-specific modifications, mixed backbonesand various reporter groups. The reaction is quick, simple, amen-able to large scale synthesis, and highly efficient. It is a very flex-ible procedure that can be used with a diverse range of alkyne andazide-labeled oligonucleotides, which are accessible from com-mercial sources. It creates novel chemical linkages that cannotbe produced by enzymatic methods. Click ligation is thereforean important addition to RNA chemistry and biochemistry.

Two different strategies were employed in this study, althoughothers could also have been used (27, 34, 35). One variant of theclick reaction (trans-ligation) was carried out between the nucleo-bases with self-templating, and also by splint-mediated ligation.The resultant synthetic hairpin ribozymes contain multiple fluor-escent labels which can be used to monitor RNA folding andmelting. The hammerhead ribozyme was constructed by an alter-native strategy, templated cis-ligation, to produce a novel triazolenucleic acid backbone mimic that is compatible with catalyticactivity. The chemistry described here could be applied to thesynthesis of many important RNAmolecules such as riboswitches(9, 10, 36), siRNA delivery systems (37), multivalent aptamers(38), and components of ribosomes (39). Using click ligation,several variants of segments of such RNA constructs could beprepared on a large scale, purified and ligated in different com-binations to provide a number of full-length RNA analoguesfor structural and biochemical studies. It is also interesting tocontemplate the possible advantages of using chemical and enzy-matic RNA ligation in tandem for the synthesis of RNA con-structs beyond the size limit of either individual method. Thiscould be achieved using oligonucleotide building blocks functio-nalized with 5′-phosphates and alkynes/azides. The enzymaticligations could be carried out first, then subsequent introductionof Cu(I) and additional alkyne/azide oligonucleotides to the mix-ture would trigger a set of orthogonal click ligation reactions. Thisstrategy could be used to place native and modified (nonhydro-lyzable) linkages at specific positions in the final construct.

Materials and MethodsMass spectra of ribozymes were recorded on a Bruker micrOTOF™ II focusESI-TOF MS instrument in ES− mode and data were processed using MaxEnt.

Sterile plastic/glassware and buffers were used for all procedures involvingRNA analogues.

Assembly of hairpin ribozymes 98-RNA, 98-DNA/RNA and 77-RNA. The threesegments of each ribozyme (Fig. 1) (10 nmol of each) in 0.2 M NaCl(950 μL) were annealed by heating at 80 °C for 5 min, cooling slowly to roomtemperature (1 h) then maintaining the temperature at 0 °C for 15 min. Asolution of CuI click catalyst was prepared from trishydroxypropyltriazoleligand 5 (Fig. 3) (28) (3.5 μmol in 0.2 M NaCl, 50.0 μL), sodium ascorbate(50.0 μmol in 0.2 M NaCl, 50.0 μL), and CuSO4 · 5H2O (0.5 μmol in 0.2 M NaCl,5.0 μL). This solution was added to the annealed ribozyme segments and thereaction mixture was kept at 0 °C for 15 min, then at room temperature for1 h. A NAP-10 gel-filtration column was used to remove reagents. The clickedhairpin ribozymes were analyzed and purified by denaturing 8% polyacryla-mide gel electrophoresis. To synthesize the short hairpin ribozyme (77-RNA)two complementary 24-mer DNA splints (10 nmol of each) (Table S1), wereadded and the reaction was carried out under the above conditions.

Templated assembly of triazole hammerhead ribozyme (Fig. 6A). Alkyne ham-merhead segment, azide hammerhead segment and hammerhead splint(Fig. 2 and Table S1), (10 nmol of each) in 0.2 M NaCl (200 μL) were annealedby heating at 80 °C for 5min and cooling slowly to room temperature (1 h). Toa solution of trishydroxypropyltriazole ligand 5 (28) (0.7 μmol) in 0.2 M NaCl(50.0 μL) was added sodium ascorbate (1.0 μmol in 0.2 M NaCl, 2.0 μL)followed by CuSO4 · 5H2O (0.1 μmol in 0.2 M NaCl, 1.0 μL) under argon. Thismixture was added to the annealed oligonucleotides and the reaction wasleft under argon at room temperature for 1 h, made up to 1 mL with waterand gel-filtered to remove reagents (NAP-10). The ligated oligonucleotideswere analyzed using denaturing 20% polyacrylamide gel electrophoresis andpurified by reversed-phase HPLC.

Cleavage of substrate with native and clicked hammerhead ribozymes (Fig. 6 Band C). The native and clicked hammerhead ribozymes (0.2 nmole of each)were dissolved in 30 μL Tris-HCl buffer (50 mM Tris-HCl, 10 mM MgCl2, pH7.6) and 0.1 nmole of the fluorescent hammerhead RNA substrate in30 μL of the same buffer was added to each ribozyme. The reaction mixtureswere incubated at 37 °C for 30 min after which time half the sample (30 μL)was removed, mixed with formamide (30 μL) and frozen in liquid nitrogen.After a further 30 min the remaining 30 μL of the reaction mixture was trea-ted in the same way. The samples were then analyzed by 20% denaturingpolyacrylamide gel electrophoresis with fluorescent detection. Cleavage ofthe nonfluorescent hammerhead substrate was carried out in the sameway using 1.25 nmole of the ribozymes and 1.0 nmole of the substrate.The reaction was analyzed after 1 h and the gel was visualized by UVshadowing.

Cleavage of substrate with 98-RNA, 98-DNA/RNA and 77-RNA hairpin ribozymes.The clicked hairpin ribozymes (0.15 nmol of each) and substrate (0.75 nmol)were dissolved in 40 μL Tris-HCl buffer (50 mM Tris-HCl, 10 mMMgCl2, pH 7.6),vortexed, and heated in a thermal cycler (PCR machine) at 55 °C for 30 minthen 5 cycles of 95 °C for 2 min and 55 °C for 30 min. Formamide (40 μL) wasadded and the reaction mixture was heated at 80 °C for 5 min to denaturethe complex, cooled on ice and analyzed by 20% denaturing polyacrylamidegel electrophoresis. To cleave the substrate for analysis by mass spectrometrythe clicked hairpin ribozyme and substrate (1.0 nmol of each) were dissolvedin 80 μL of Tris-HCl buffer and heated at 55 °C for 1 h, after which the solutionwas made up to 1 mL with water and desalted by NAP-10 gel-filtration.

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