Optimized methods for the enzymatic ligation of oligoribonucleotides and analogues

The preparation of oligoribonucleotides is of great interest for structural and functional investigations in biomolecular chemistry and biology. Relevant RNAs are relatively long and contain often modifications. Two methods for their preparation are currently available: automated chemical synthesis and in vitro transcription. The chemical synthesis allows the preparation of relatively short (< 60mers) pure RNA sequences containing modified nucleotides. In contrast, in vitro transcription is appropriate for the synthesis of long RNA sequences but does not allow the incorporation of modifications. An elegant solution to this dilemma is offered by enzymatic ligations of RNA fragments with T4-DNA ligase, which is template-dependent and therefore highly selective. Here, we present a comprehensive study of parameters, which govern the effectiveness of this ligation. The influence of secondary structures present within templates were studied in detail. That allowed to design a new type of hybrid template, containing both DNA and 2'-OMeRNA nucleotides, which were particularly efficient (Chapter 3.2). Ligation reactions with structured substrates and different types of oligonucleotides were carried out, leading to a better comprehension of the enzyme mechanism. In this context, we found very strong indications for a different recognition of the donor and acceptor substrates by the enzyme (Chapter 3.2 and 3.3). Optimization of the enzymatic ligations with different substrates or templates has showed the importance of secondary structures for design of a successful reaction (Chapter 3.2 and 3.3). The lessons drawn from these preliminary experiments have been employed for the reliable preparative synthesis of natural oligoribonucleotides containing modifications (Chapter 3.4) and the preparation of long RNA sequences (Chapter 3.5).


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