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

Abstract

The CuAAC reaction (click chemistry) has been used in conjunction with solid-phase synthesis to produce catalytically active hairpin ribozymes around 100 nucleotides in length. Cross-strand ligation through neighboring nucleobases was successful in covalently linking presynthesized RNA strands with high efficiency (transligation). In an alternative strategy, intrastrand click ligation was employed to produce a functional hammerhead ribozyme containing a novel nucleic acid backbone mimic at the catalytic site (cisligation). The ability to synthesize long RNA strands by a combination of solid-phase synthesis and click ligation is an important addition to RNA chemistry. It is compatible with a plethora of sitespecific modifications and is applicable to the synthesis of many biologically important RNA molecules.ligonucleotide chemistry is central to the advancement of core technologies such as DNA sequencing and genetic analysis and has impacted greatly on the discipline of molecular biology (1, 2). Oligonucleotides and their analogues are essential tools in these areas. They are produced by automated solid-phase phosphoramidite synthesis, a highly efficient process that can be used to assemble DNA strands over 100 bases in length (3). Synthesis of RNA is less efficient owing to problems caused by the presence of the 2′-hydroxyl group of ribose, which requires selective protection during oligonucleotide assembly. This reduces the coupling efficiency of RNA phosphoramidite monomers due to steric hindrance. In addition, side-reactions that occur during the removal (or premature loss) of the 2′-protecting groups cause phosphodiester backbone cleavage and 3′ to 2′ phosphate migration (4). Although several ingenious strategies have been developed to minimize these problems (5) and to improve the synthesis of long RNA (6), the chemical complexity of solid-phase RNA synthesis dictates that constructs longer than 50 nucleotides in length remain difficult to prepare. Most biologically important RNA molecules such as ribozymes (7), aptamers (8), and riboswitches (9, 10) are significantly longer than this, so new approaches to the synthesis of RNA analogues are urgently required. Although RNA synthesis by transcription might seem a viable alternative, it does not permit the site-specific incorporation of multiple modifications at sugars, bases, or phosphates. In contrast, automated solid-phase RNA synthesis is compatible with the introduction of fluorescent tags, isotopic labels (for NMR studies) and other groups to improve biological activity and resistance to enzymatic degradation. The scope and utility of important RNA constructs can be significantly extended by such chemical modifications, and the scale of chemical synthesis is potentially unlimited (11). These are important factors when planning structural studies on RNA or the development of therapeutic oligoribonucleotides analogues, and should be considered when devising new methods of RNA assembly. Enzymatic ligation might appear to be a method of achieving some of th...

Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli

“…The functionalized resin required for the solid-phase synthesis of oligonucleotides terminating with 3′-propargyl Me dC (cytosine equivalent, Fig. 2) was prepared from thymidine as previously described (26). A polystyrene support was used in this case, to achieve high coupling yields and produce 100-mer oligonucleotides of the purity required for efficient click ligation.…”

A triazole linkage that mimics the DNA phosphodiester group in living systems

“…We have also carried out similar work on RNA (El-Sagheer & Brown, 2010), and this is a fruitful area for future research. The chemical ligation approach is not necessarily limited to the linker reported here; alternative chemical reactions and backbone linkages might also be suitable for this purpose.…”

In vitro circularization of RNA

“…Another option is the reaction of the unique primary alcohol at the 5 0 -end with triphenylphosphonium iodide to generate a good leaving group and thus making the 5 0 -terminus suitable for reaction with a nucleophile, as for example an azide. 23,24 The azide may be directly used for Click cyclization (see below) or, upon reduction to an amino group, for conjugation with a desired functionality via peptide chemistry. 25 Specific 5 0 -RNA functionalization can be also achieved by transcription priming, adding to the transcription mixture an initiator nucleotide that carries no triphosphate for polymerization and therefore can be used only for priming RNA synthesis at the 5 0 -end.…”