Acide ribonucléique de transfert

Les acides ribonucléiques de transfert, ou ARN de transfert ou ARNt, sont de courts ARN, longs de 75 à 95 nucléotides, qui interviennent lors de la synthèse des protéines dans la cellule. Ce sont des intermédiaires clés dans la traduction du message génétique et dans la lecture du code génétique. Ils apportent les acides aminés au ribosome, la machine cellulaire responsable de l'assemblage des protéines à partir de l'information génétique contenue dans l'ARN messager. Les cellules vivantes contiennent quelques dizaines de sortes d'ARNt, chacune d'elles étant spécifique de l'un des acides aminés. Les ARNt se terminent du côté 3' par une extrémité simple-brin conservée -CCA constante. L'acide aminé est accroché par une liaison ester sur le ribose de l'adénosine terminale de cette extrémité. Description des ARN de transfert Les ARNt sont des acides ribonucléiques simple brin, longs d'environ 75 à 95 nucléotides, que l'on trouve dans le cytoplasme ou dans les organites des

Synthesis of modified nucleosides for the incorporation into tRNAs

Sébastien Porcher

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

Preparation of aminoacylated tRNAs and analogues

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

Preparation of aminoacylated tRNAs and analogues

EPFL2006

Synthesis of modified nucleosides for the incorporation into tRNAs

Sébastien Porcher

EPFL2006