New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead

Afaf 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 Science and 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 May 9, 2010)

The CuAAC reaction (click chemistry) has been used in conjunction objectives (12). T4 RNA ligase can be used to join nucleotides in with solid-phase synthesis to produce catalytically active hairpin single-stranded regions such as RNA loops, whereas T4 DNA ribozymes around 100 nucleotides in length. Cross-strand ligation ligase functions on double-stranded RNA. However, neither en- through neighboring nucleobases was successful in covalently zyme can be used to link together RNA strands at other positions, linking presynthesized RNA strands with high efficiency (trans- such as across the bases or sugars, or to create unnatural linkages. ligation). In an alternative strategy, intrastrand click ligation was The use of ligases has other drawbacks; they are often contami- employed to produce a functional hammerhead contain- nated with RNases that can partially degrade the ligation prod- ing a novel nucleic acid backbone mimic at the catalytic site (cis- ucts, and the ligation protocols are quite complicated, requiring ligation). The ability to synthesize long RNA strands by a combina- phenol extraction to remove the protein. Moreover, enzymatic tion of solid-phase synthesis and click ligation is an important ligation methods are not suitable for the large scale synthesis of addition to RNA chemistry. It is compatible with a plethora of site- RNA, and the yields of enzymatic RNA ligation are often low. This specific modifications and is applicable to the synthesis of many is partly due to the tendency of T4 RNA ligase to cyclize phos-

biologically important RNA molecules. phorylated RNA strands and to join together incorrect strands. CHEMISTRY Here we describe click RNA ligation, a strategy that overcomes chemical ligation ∣ click RNA ligation ∣ CuAAC ∣ triazole backbone ∣ the above limitations and could lead to a step-change in the synth- biocompatible esis of biologically active RNA constructs. The CuAAC reaction (13, 14) was chosen for RNA ligation owing to its very high speed, ligonucleotide chemistry is central to the advancement of efficiency, orthogonality with functional groups present in nucleic Ocore technologies such as DNA sequencing and genetic acids and compatibility with aqueous media. In this procedure, analysis and has impacted greatly on the discipline of molecular individual RNA oligonucleotides are assembled by automated

biology (1, 2). Oligonucleotides and their analogues are essential solid-phase synthesis, purified by HPLC then chemically ligated BIOCHEMISTRY tools in these areas. They are produced by automated solid-phase by click chemistry (15) to produce much larger molecules. In this phosphoramidite synthesis, a highly efficient process that can be study we demonstrate the power of click ligation by synthesising a used to assemble DNA strands over 100 bases in length (3). series of chemically modified hairpin and hammerhead ribo- Synthesis of RNA is less efficient owing to problems caused by zymes. The hairpin ribozyme belongs to a family of catalytic the presence of the 2′-hydroxyl group of ribose, which requires that cleave their RNA substrates in a reversible reaction ′ ′ ′ selective protection during oligonucleotide assembly. This to generate 2 ,3 -cyclic phosphate and 5 -hydroxyl termini. It was reduces the coupling efficiency of RNA phosphoramidite mono- discovered in tobacco ringspot virus RNA where ribo- mers due to steric hindrance. In addition, side-reactions that oc- zyme-mediated self-cleavage and ligation reactions participate cur during the removal (or premature loss) of the 2′-protecting in processing RNA replication intermediates (16). It was chosen groups cause phosphodiester backbone cleavage and 3′ to 2′ for the current study for three reasons: Its size is such that direct phosphate migration (4). Although several ingenious strategies chemical synthesis is problematic and therefore click ligation be- have been developed to minimize these problems (5) and to im- comes a valuable technique, its biophysical and catalytic proper- prove the synthesis of long RNA (6), the chemical complexity of ties have been extensively studied and are well understood (17), solid-phase RNA synthesis dictates that constructs longer than 50 and its functionality can be extended by the incorporation of che- nucleotides in length remain difficult to prepare. Most biologi- mical modifications such as fluorescent tags (18). In this study cally important RNA molecules such as ribozymes (7), aptamers three analogues of the hairpin ribozyme were synthesized from individual oligonucleotides by crosslinking the side-chains of (8), and riboswitches (9, 10) are significantly longer than this, so “ ” new approaches to the synthesis of RNA analogues are urgently modified uracil bases in opposite strands of the unclicked seg- required. Although RNA synthesis by transcription might seem a ments (trans-ligation). An RNA ribozyme (98-RNA) was synthe- viable alternative, it does not permit the site-specific incorpora- sized and a DNA/RNA chimera (98-DNA/RNA) was made in tion of multiple modifications at sugars, bases, or phosphates. In which segments a and b contain tracts of DNA and segment c contrast, automated solid-phase RNA synthesis is compatible is entirely DNA (Fig. 1, Left). Ribozymes are known to tolerate with the introduction of fluorescent tags, isotopic labels (for NMR studies) and other groups to improve biological activity Author contributions: A.H.E. and T.B. designed research, performed research, analyzed and resistance to enzymatic degradation. The scope and utility data, and wrote the paper. of important RNA constructs can be significantly extended by The authors declare no conflict of interest. such chemical modifications, and the scale of chemical synthesis This article is a PNAS Direct Submission. P.H.S. is a guest editor invited by the is potentially unlimited (11). These are important factors when Editorial Board. planning structural studies on RNA or the development of ther- Freely available online through the PNAS open access option. apeutic oligoribonucleotides analogues, and should be consid- 1To whom correspondence should be addressed. E-mail: [email protected]. ered when devising new methods of RNA assembly. Enzymatic This article contains supporting information online at www.pnas.org/lookup/suppl/ ligation might appear to be a method of achieving some of these doi:10.1073/pnas.1006447107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1006447107 PNAS Early Edition ∣ 1of6 Downloaded by guest on October 1, 2021 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 ribozyme products 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 RNA hairpin ribozyme 98-RNA and DNA–RNA hybrid 98-DNA/RNA. The sequences are the same but 98-DNA/RNA contains deoxyribose sugars at the positions marked 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 spectrum of 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).

deoxyribonucleosides outside the catalytic site and the incorpora- analogues (25). Oligonucleotide sequences are shown in Figs. 1 tion of DNA offers the possibility of synthesising longer fragments and 2 and in Table S1. For the trans-click ligation reactions, the for click ligation. Therefore we were anxious to confirm that individual oligonucleotide segments require an alkyne or azide the CuAAC reaction is compatible with DNA–RNA hybrids. A group at the 5-position of a uracil base at the appropriate locus. shorter 77-nucleotide version of the hairpin ribozyme was also prepared (77-RNA, Fig. 1, Right) to evaluate a simple splint- mediated modification of the above click ligation strategy. In a different approach, a (19, 20) was made by the splint-mediated cis-ligation of two RNA strands (Fig. 2). The hammerhead is a small catalytic RNA motif that is found in plant RNA viruses, satellite RNA, viroids, and repetitive satellite DNA (21) where it catalyzes cleavage and ligation of RNA during rolling circle replication (22, 23). It was chosen as a model to in- vestigate the chemistry of a new triazole nucleic acid backbone mimic in click ligation, and to explore its biocompatibility. Results and Discussion Hairpin Ribozymes. The oligonucleotide building blocks required in the assembly of the hairpin ribozyme analogues (segments a, b, and c in Fig. 1) were prepared by automated solid-phase phosphoramidite synthesis using 2′-t-butyldimethylsilyl (TBS) Fig. 2. Structure and sequence of the hammerhead ribozyme (green) and protected monomers for the RNA tracts. The TBS method of substrate (blue) with cleavage site in red. The triazole linkage lies in the cat- RNA synthesis (24) was chosen as it is widely used, the requisite alytic pocket opposite to the cleavage site between C3 and U4. Nucleotides reagents are readily available, and it has been shown to be effec- C3 and G8 form a in the active conformation of the ribozyme– tive in the synthesis of a wide range of chemically modified RNA substrate complex (33).

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1006447107 El-Sagheer and Brown Downloaded by guest on October 1, 2021 The reaction scheme and the structures of the alkyne and azide- necessary. For the smaller hairpin ribozyme 77-RNA we elected labeled nucleoside components 3 and 4 are shown in Fig. 3. to explore the use of two complementary 24-mer DNA splints to Azides are unstable to PIII, and do not survive the conditions hold the short arms of the ribozyme segments in place during of phosphoramidite oligonucleotide synthesis. Therefore, the click ligation. This method gave a clean reaction and the resultant azide functionalities were introduced after oligonucleotide as- short hairpin ribozyme was purified by polyacrylamide gel- sembly by selective reaction of the N-hydroxysuccinimide ester electrophoresis. of azidohexanoic acid 2 with the primary amino groups of the The catalytic activity of the three hairpin ribozyme analogues modified uracil bases (26). All oligonucleotides were purified was investigated by running cleavage reactions at 55 °C in the by reversed-phase HPLC and analyzed by mass spectrometry presence of five equivalents of the RNA substrate (Fig. 4). In prior to click ligation. For construction of the all-RNA and all cases five cycles of denaturation (95 °C) followed by cleavage DNA/RNA 98-mer ribozymes the individual segments were an- (55 °C) consumed the substrate. It was cleaved at the expected nealed and covalently linked in a self-templated CuAAC reaction site (indicated in Fig. 1), as confirmed by mass spectrometry (27). The CuI catalyst was generated in situ from CuII sulfate and of the two fragments (ES−, found: 5944 Da, 2154 Da, requires: aqueous sodium ascorbate, and a water-soluble CuI-binding 5943 Da, 2155 Da). The ribozymes also cleaved the substrate ligand 5 was added to prevent RNA degradation (28). During without temperature cycling (Fig. S3). The clicked ribozymes the reaction segment a was crosslinked to segment b near termi- were robust and could be isolated from the cleavage gels and re- nus B, and segment b to segment c near terminus C across the used in subsequent reactions with no significant loss of activity. major grooves of the ribozyme stems. The crosslinking positions The fact that the triazole hairpin ribozyme cleaves its substrate are indicated in Fig. 1, and the chemical structure of the resultant rapidly and produces the same products as the natural ribozyme triazole linkage 6 is shown in Fig. 3. The two simultaneous click is not surprising as the click linkages are located remotely from ligation reactions proceeded efficiently in the assembly of both the active site. This design feature can be conveniently incorpo- ribozymes, and full-length constructs were purified by polyacry- rated into most large RNA constructs. This click linkage stretches lamide gel-electrophoresis (Fig. 1, Left). Despite the self-templat- across the major groove where it does not perturb the duplex or ing, a minor side-reaction occurred due to cyclization of segment change its conformation (26). Cross-strand linking between nu- b, which contains both alkyne and azide. This impurity was easily cleobases in RNA is unique to chemical ligation and cannot removed during ribozyme purification. A shorter 77 nucleotide be achieved by enzymatic methods.

version of the hairpin ribozyme was also prepared (77-RNA, CHEMISTRY Fig. 1, Right), but in this case segment b was synthesized with The Hammerhead Ribozyme. A minimal hammerhead ribozyme two azide groups, one near the 5′- terminus and the other near was synthesized by splint-mediated intrastrand click ligation the 3′-end, rather than alkyne and azide. Segments a and c were (cis- ligation) of 3′-alkyne and 5′-azide oligoribonucleotides. This synthesized with an alkyne near their 3′- and 5′-ends respectively alternative method was chosen to investigate the suitability of to ensure chemical complementarity with segment b. This facile click chemistry for linking RNA strands through their backbone change in strategy eliminated the possibility of unwanted cycliza- rather than between the nucleobases. The success of this strategy tion of segment b and is an example of the flexibility offered by in producing an active ribozyme requires the design of a triazole chemical ligation. Segments a, b, and c were labeled with cy5, cy3, linkage that closely resembles a phosphodiester group. The struc- and FAM respectively to facilitate fluorescence resonance energy ture of this linkage and the synthetic strategy to produce it are BIOCHEMISTRY transfer (FRET) studies (7, 29). There are many cases when RNA shown in Fig. 5. The key building block for the synthesis of assembly cannot be self-templated, so an alternative strategy is the 3′-alkyne labeled RNA strand is 5′-DMT-3′-propargyl-5- methyldeoxycytidine 9. This was made in two steps, the first involving 3′-alkylation of 5′-DMT thymidine 7 with propargyl bromide 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 to 5-methyl cytosine was carried out using phosphorus oxychloride, N-methylimidazole and aqueous ammonia. Compound 9 was coupled to succinylated aminoalkyl solid support to give deriva- tized support 10, which was used in the solid-phase synthesis of 3′-propargyl oligonucleotide 11 (Fig. 5). This solid support will be useful in the synthesis of oligonucleotides for various click liga- tion or labeling applications. The 5′-azide RNA strand 13 was made from the corresponding unmodified 5′-hydroxyl RNA con- gener 12 by treatment of the support-bound oligonucleotide with methyltriphenoxyphosphonium iodide followed by sodium azide. This 5′-azide labeling method has been used previously on DNA

Fig. 4. Hairpin ribozyme substrate cleavage reactions. Lanes 1, 3, 5: cleavage reactions of 98-RNA, 98-DNA/RNA and 77-RNA ribozymes respectively with 5 Fig. 3. The synthetic procedure used to covalently link the arms of the hair- equivalents of substrate. Lanes 2, 4, 6: control—uncleaved substrate. 20% pin ribozyme across the major groove of the duplex. polyacrylamide gels visualized by fluorescence.

El-Sagheer and Brown PNAS Early Edition ∣ 3of6 Downloaded by guest on October 1, 2021 Fig. 5. Synthesis of the hammerhead ribozyme. Synthesis of alkyne and azide RNA strands, construction hammerhead, and structure of triazole backbone.

(31) and here we demonstrate its compatibility with RNA. The firms that in this structure the designed linkage is a suitable two components of the hammerhead ribozyme (11 and 13) were analogue of a phosphodiester group. designed with extra thymidine nucleotides at the termini to aid To further demonstrate that the triazole linkage is a viable gel-filtration, whereas the 5′-FAM dye on the DNA splint was substitute for a phosphodiester group we carried out UV-melting included to differentiate it from the clicked ribozyme during re- versed-phase HPLC purification. Splint-mediated ligation of the two RNA strands 11 and 13 proceeded smoothly to give ribozyme 14 with a triazole linkage in the backbone (Fig. 6A, lane 4). The modified hammerhead ribozyme was characterized by mass spec- trometry (ES-, found: 7585 Da, requires: 7585 Da) and was shown to cleave its substrate, generating an identical product to that formed by the normal unmodified ribozyme (Fig. 6B, lanes 1 and 2). Cleavage occurred at the predicted scissile phosphodiester bond 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- Fig. 6. (A). CuAAC reaction to synthesize the hammerhead ribozyme, 20% escein labeled substrate demonstrated complete cleavage in polyacrylamide gel: Lanes 1, 2: starting oligonucleotides (alkyne and azide), 30 min at 37 °C, similar to the native ribozyme (Fig. 6C). This is lane 3: DNA splint, lane 4: crude reaction mixture. (B). Hammerhead ribozyme a satisfying result, particularly as the unnatural triazole linkage cleavage reaction with nonfluorescent substrate. Lane 1: S þ TR, lane 2: is located at the active site between residues C3 and U4 of the S þ NR, lane 3: TR, lane: 4 NR, lane 5: S. (S ¼ uncleaved substrate, ¼ ¼ ribozyme (Fig. 2). X-ray structures of the minimal (19, 20, 32) NR normal ribozyme control, TR triazole ribozyme). Gels A and B visua- lized by UV shadowing. TR (24-mer) is longer than NR (16-mer) due to the and full-length hammerhead ribozymes (33) confirm the critical additional thymidines at the termini. (C). Hammerhead ribozyme cleavage positioning of C3. It is postulated that a base pair between C3 reaction with 5′-fluorescein labeled substrate. Lane 1: FS, lane 2: FS þ NR and G8 holds the ribozyme in its catalytically competent confor- 30 min, lane 3: FS þ TR 30 min, lane 4: FS, lane 5: FS þ NR 1 h, lane 6: mation (33). The observed activity of the triazole ribozyme con- FS þ TR 1 h. (FS ¼ fluorescent substrate, gel imaged by fluorescence).

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1006447107 El-Sagheer and Brown Downloaded by guest on October 1, 2021 studies on the natural and triazole ribozymes with their RNA sub- Sterile plastic/glassware and buffers were used for all procedures involving strate in the absence of magnesium ions (to prevent substrate RNA analogues. cleavage). We obtained melting curves that were very similar (Fig. S4), with a slight lowering in melting temperature in the Assembly of hairpin ribozymes 98-RNA, 98-DNA/RNA and 77-RNA. The three segments of each ribozyme (Fig. 1) (10 nmol of each) in 0.2 M NaCl triazole case (ΔTm ¼ 1.1 °C). We then hybridized the native μ and triazole hammerhead ribozymes to their DNA complement (950 L) were annealed by heating at 80 °C for 5 min, cooling slowly to room – temperature (1 h) then maintaining the temperature at 0 °C for 15 min. A to produce DNA RNA hybrid duplexes. In this case the triazole I analogue was almost as stable as its natural counterpart solution of Cu click catalyst was prepared from trishydroxypropyltriazole ΔT ¼ 3 8 ligand 5 (Fig. 3) (28) (3.5 μmol in 0.2 M NaCl, 50.0 μL), sodium ascorbate ( m . °C) (Fig. S4). The CD spectra of the above constructs μ μ 5 μ indicate that the triazole modification does not change the duplex (50.0 mol in 0.2 M NaCl, 50.0 L), and CuSO4 · H2O(0.5 mol in 0.2 M NaCl, 5.0 μL). This solution was added to the annealed ribozyme segments and the conformation (Fig. S5). Although it would not generally be reaction mixture was kept at 0 °C for 15 min, then at room temperature for necessary to place the triazole modification at a critical catalytic 1 h. A NAP-10 gel-filtration column was used to remove reagents. The clicked position when designing RNA constructs we have shown here hairpin ribozymes were analyzed and purified by denaturing 8% polyacryla- that such a substitution can lead to stable biologically active mide gel electrophoresis. To synthesize the short hairpin ribozyme (77-RNA) ribozymes, thereby demonstrating the biocompatibility of the two complementary 24-mer DNA splints (10 nmol of each) (Table S1), were triazole linkage. added and the reaction was carried out under the above conditions. Conclusion Templated assembly of triazole hammerhead ribozyme (Fig. 6A). Alkyne ham- In this study click chemistry has been used to ligate presynthe- merhead segment, azide hammerhead segment and hammerhead splint sized oligonucleotides to construct RNA molecules and DNA/ (Fig. 2 and Table S1), (10 nmol of each) in 0.2 M NaCl (200 μL) were annealed RNA chimeras up to 100 nucleotides in length. The method is by heating at 80 °C for 5 min and cooling slowly to room temperature (1 h). To compatible with site-specific modifications, mixed backbones a solution of trishydroxypropyltriazole ligand 5 (28) (0.7 μmol) in 0.2 M NaCl and various reporter groups. The reaction is quick, simple, amen- (50.0 μL) was added sodium ascorbate (1.0 μmol in 0.2 M NaCl, 2.0 μL) able to large scale synthesis, and highly efficient. It is a very flex- followed by CuSO4 · 5H2O(0.1μmol in 0.2 M NaCl, 1.0 μL) under argon. This ible procedure that can be used with a diverse range of alkyne and mixture was added to the annealed oligonucleotides and the reaction was azide-labeled oligonucleotides, which are accessible from com- left under argon at room temperature for 1 h, made up to 1 mL with water mercial sources. It creates novel chemical linkages that cannot and gel-filtered to remove reagents (NAP-10). The ligated oligonucleotides

be produced by enzymatic methods. Click ligation is therefore were analyzed using denaturing 20% polyacrylamide gel electrophoresis and CHEMISTRY an important addition to RNA chemistry and biochemistry. purified by reversed-phase HPLC. Two different strategies were employed in this study, although others could also have been used (27, 34, 35). One variant of the Cleavage of substrate with native and clicked hammerhead ribozymes (Fig. 6 B click reaction (trans-ligation) was carried out between the nucleo- and C). The native and clicked hammerhead ribozymes (0.2 nmole of each) μ bases with self-templating, and also by splint-mediated ligation. were dissolved in 30 L Tris-HCl buffer (50 mM Tris-HCl, 10 mM MgCl2,pH The resultant synthetic hairpin ribozymes contain multiple fluor- 7.6) and 0.1 nmole of the fluorescent hammerhead RNA substrate in escent labels which can be used to monitor RNA folding and 30 μL of the same buffer was added to each ribozyme. The reaction mixtures μ melting. The hammerhead ribozyme was constructed by an alter- were incubated at 37 °C for 30 min after which time half the sample (30 L) μ BIOCHEMISTRY native strategy, templated cis-ligation, to produce a novel triazole was removed, mixed with formamide (30 L) and frozen in liquid nitrogen. μ nucleic acid backbone mimic that is compatible with catalytic After a further 30 min the remaining 30 L of the reaction mixture was trea- activity. The chemistry described here could be applied to the ted in the same way. The samples were then analyzed by 20% denaturing synthesis of many important RNA molecules such as riboswitches polyacrylamide gel electrophoresis with fluorescent detection. Cleavage of (9, 10, 36), siRNA delivery systems (37), multivalent aptamers the nonfluorescent hammerhead substrate was carried out in the same way using 1.25 nmole of the ribozymes and 1.0 nmole of the substrate. (38), and components of ribosomes (39). Using click ligation, The reaction was analyzed after 1 h and the gel was visualized by UV several variants of segments of such RNA constructs could be shadowing. prepared on a large scale, purified and ligated in different com- binations to provide a number of full-length RNA analogues Cleavage of substrate with 98-RNA, 98-DNA/RNA and 77-RNA hairpin ribozymes. for structural and biochemical studies. It is also interesting to The clicked hairpin ribozymes (0.15 nmol of each) and substrate (0.75 nmol) contemplate the possible advantages of using chemical and enzy- were dissolved in 40 μL Tris-HCl buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 7.6), matic RNA ligation in tandem for the synthesis of RNA con- vortexed, and heated in a thermal cycler (PCR machine) at 55 °C for 30 min structs beyond the size limit of either individual method. This then 5 cycles of 95 °C for 2 min and 55 °C for 30 min. Formamide (40 μL) was could be achieved using oligonucleotide building blocks functio- added and the reaction mixture was heated at 80 °C for 5 min to denature nalized with 5′-phosphates and alkynes/azides. The enzymatic the complex, cooled on ice and analyzed by 20% denaturing polyacrylamide ligations could be carried out first, then subsequent introduction gel electrophoresis. To cleave the substrate for analysis by mass spectrometry of Cu(I) and additional alkyne/azide oligonucleotides to the mix- the clicked hairpin ribozyme and substrate (1.0 nmol of each) were dissolved ture would trigger a set of orthogonal click ligation reactions. This in 80 μL of Tris-HCl buffer and heated at 55 °C for 1 h, after which the solution strategy could be used to place native and modified (nonhydro- was made up to 1 mL with water and desalted by NAP-10 gel-filtration. lyzable) linkages at specific positions in the final construct. ACKNOWLEDGMENTS. We thank Professor D.M.J. Lilley for helpful discussions. Materials and Methods The research leading to these results has received funding from the European Mass spectra of ribozymes were recorded on a Bruker micrOTOF™ II focus Community’s Seventh Framework Programme (FP7/2007-2013) under Grant ESI-TOF MS instrument in ES− mode and data were processed using MaxEnt. HEALTH-F4-2008-201418) entitled READNA.

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