Optimized methods for the enzymatic ligation of oligoribonucleotides and analogues

Frédéric David Meylan
EPFL, 2006
Thèse EPFL

Résumé

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).

Official source

https://infoscience.epfl.ch/record/89650?ln=fr

À propos de ce résultat

Cette page est générée automatiquement et peut contenir des informations qui ne sont pas correctes, complètes, à jour ou pertinentes par rapport à votre recherche. Il en va de même pour toutes les autres pages de ce site. Veillez à vérifier les informations auprès des sources officielles de l'EPFL.

Concepts associés

Chargement

Publications associées

Chargement

Frédéric David Meylan
EPFL, 2006
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/89650?ln=fr

À propos de ce résultat

Concepts associés (25)

Concepts associés

Chargement

Publications associées (24)

Publications associées

Chargement

More than 100 modified nucleosides have been found in naturally occurring RNA sequences. These modifications are involved in the correct folding, in the maintenance of the reading frame during translation and in the proper interaction with enzymes and protein complexes. For an exhaustive study of their function it would be necessary to incorporate by chemical synthesis such modifications at selected positions of RNA sequences. Here we present optimized protocols for the preparation of a large variety of 2'-O-TOM protected ribonucleoside phosphoramidite building blocks containing the most frequently encountered modified nucleobases m2G, m22G, m1A, m5U, D, m5C, ψ, m1I, i6A, m62A, m6A, m1G, t6A, I and imG. In addition, the non-natural ribonucleoside phosphoramidites containing isoG, isoC and 4-desmethyl-5-methylwyosine have been prepared (Chapters I, II and III). For the introduction of base-labile modifications into RNA sequences, modified deprotection conditions in combination with the new N2-methoxyacetyl protected guanosine phosphoramidite have been developed (Chapter V). For the first time, the sensitive modified nucleoside wyosine was incorporated into a 18mer RNA sequence and into the anticodon loop of a truncated tRNA. For this purpose, enzymatic ligation strategies, based on T4 RNA and T4 DNA ligase, were evaluated and optimized (Chapters III and V). The decoding properties of modified anticodons have been reviewed and new models, based on the formation of secondary hydrogen bonds have been proposed (Chapter IV). For the efficient preparation of tRNA analogues esterified with unnatural amino acids, a synthesis of modified RNA sequences containing a 3'-terminal 2'-deoxy-2'-thioadenosine was developed. In analogy to the "native chemical ligation" of oligopeptides, its spontaneous and site-specific aminoacylation with weakly activated amino acid thioesters occurred efficiently in buffered aqueous solutions and under a wide range of conditions. This concept could be employed for a straightforward aminoacylation of analogously modified tRNAs (Chapter VI).

EPFL2006

Chargement

By automated synthesis, we prepd. hybrid oligonucleotides consisting of covalently linked RNA and p-DNA sequences (p-DNA = 3'-deoxyribopyranose (4'->2')-oligonucleotides). The pairing properties of corresponding hybrid duplexes formed from fully complementary single strands were investigated. An uninterrupted p-p-stacking at the p-DNA/RNA interface and cooperative pairing between the two systems was achieved by connecting them via a 4'-p-DNA-2'->5'-RNA-3' and 5'-RNA-2'->4'-p-DNA-2' phosphodiester linkage, resp. The RNA 2'-phosphoramidites required for the formation of the RNA-2'->4'-p-DNA phosphodiester linkage were synthesized from the corresponding, 3'-O-tom-protected ribonucleosides (tom = [(triisopropylsilyl)oxy]methyl). Analogs of the FMN binding aptamer and the hammerhead ribozyme were prepd. Each of these analogs consisted of two p-DNA/RNA hybrid single strands with complementary p-DNA sequences, designed to substitute stem/loop and stem motifs within the parent compds. By comparative binding and cleavage studies, it was found that mixing of the two complementary p-DNA/RNA hybrid sequences resulted in the formation of the fully functional analogs of the FMN-binding aptamer and of the hammerhead ribozyme, resp. [on SciFinder (R)]

2002

Chargement

The ribosome-mediated, site-specific incorporation of unnatural amino acids into proteins is a powerful tool for creating protein analogues with specific steric, chemical, electronic or spectroscopic properties, in vitro and in vivo. The required aminoacylated tRNA analogues are prepared semisynthetically, by ribozyme-catalyzed amino acid transfer from short aminoacylated RNA sequences, from complementary PNA amino acid thioesters or by modified aminoacyl-tRNA synthetases. The common semisynthetic preparation of aminoacylated tRNA analogues is based on the ligation of aminoacylated dimers with a truncated tRNA, produced by run-off transcription. However, the presence of overtranscripts and the lability of the amino acid/nucleotide ester bond represent a severe limitation in terms of purity and efficiency of this ligation reaction. In this context, we first have optimized the in vitro RNA transcription reaction to obtain a straightforward access to homogeneous truncated tRNAs. We found that addition of 3.5 equivalents of GMP results not only in the selective formation of transcripts with 5'-monophosphate termini, but also in the formation of less overtranscripts without affecting the yield. Moreover, we have investigated the purification of in vitro transcription reaction mixtures by anion exchange HPLC, resulting in a simple and efficient isolation/purification protocol (Chapter 2.1). We have prepared tRNAs aminoacylated with L-lysine -BODIPY and L-lysine-fluorescein conjugates, respectively, by efficient blunt-end ligation of a corresponding, protected and stabilized aminoacylated dimer with truncated tRNAs. The resulting protected and stabilized aminoacylated tRNAs could be characterized by ESI-MS. The presence of two photolabile groups at the 3'-terminal 2'-OH group and on the α-amino group of the amino acid allowed an efficient preparation and purification of the aminoacylated tRNA analogues and to store them for an unlimited time at -20° (Chapter 2.2). Although RNA transcription is an easy method to prepare truncated tRNAs, this method suffers from severe limitations to introduce modified nucleotides into tRNAs. To overcome this limitation, we have optimized their preparation from synthetic RNA fragments by T4-DNA ligase mediated ligation reactions. An amber suppressor tRNA derived from the E.coli tRNAPhe was used as model system and a ligation from three RNA fragments was investigated. Specifically, the design of the templates and the reaction conditions were evaluated and optimized. We found that 2'-OMeRNA templates or DNA/2'-OMeRNA hybrid templates could lead to a significantly better conversion than the commonly employed DNA templates. Under optimized conditions, the full tRNA could be assembled with an efficiency of 65% (Chapter 2.3). Finally within the context of a new concept for tRNA aminoacylation, we have prepared a biotinyl-lysine tRNA containing a 3'-terminal 2'-deoxy-2'-thioadenosine. The new concept was developed in our group by S. Porcher and M. Meyyappan, who found that such RNA analogues react efficiently, spontaneously and site-specifically with weakly activated amino acid thioesters in buffered aqueous solutions and under a wide range of conditions (Chapter 2.4). Some efforts towards the development of translation assays are described. Unfortunately, the corresponding experiments were not successful (Chapter 2.5).

EPFL2006

Chargement

EPFL2006