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New Strategy for the Synthesis of Chemically Modified RNA Constructs Exemplified by Hairpin and Hammerhead Ribozymes

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New Strategy for the Synthesis of Chemically Modified RNA Constructs Exemplified by Hairpin and Hammerhead Ribozymes New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes 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 ribozyme 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 RNAs 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 satellite 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 hammerhead ribozyme (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)
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