RNA-sequence or nucleoside/nucleotide analogues and method for producing the analogues

Nucleic acid sequence analogues, in particular tRNA analogues comprising in the place of a 3'-terminal adenosine a 3'-terminal 2'-deoxy-2'-thioadenosine are able to react site-specifically with weakly activated amino acid derivatives (e.g. phenylthioesters of amino acids), thereby connecting the amino acid to the 2'-thiol-group of the 3'-terminal thioadenosine via a thioester bond. A fast and thermodynamically favored intramolecular transesterification reaction leads then to the formation of the corresponding 3'-O-acyl-derivative. A correspondingly modified tRNA is an active analogue of an aminoacylated tRNA and allows introduction of the amino acid bound thereto into proteins. The aminoacylated tRNA analogue according to the invention can be isolated in pure form under acidic conditions (pH 5.0). At pH 7.4 and 25 DEG C it is found to have a half-life of 25 min, which compares well with the half-life of 40 min for the corresponding native compound.

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

Synthesis of 2'-O-[(triisopropylsilyl)oxy]methyl(=tom)-protected ribonucleoside phosphoramidites containing various nucleobase analogs

Sébastien Porcher

The first results of a study aiming at an efficient prepn. of a large variety of 2'-O-[(triisopropylsilyl)oxy]methyl(=tom)-protected ribonucleoside phosphoramidite building blocks contg. modified nucleobases are reported. All of the here presented nucleosides have already been incorporated into RNA sequences by several other groups, employing 2'-O-tbdms- or 2'-O-tom-protected phosphoramidite building blocks (tbdms=(tert-butyl)dimethyl-silyl). We now optimized existing reactions, developed some new and shorter synthetic strategies, and sometimes introduced other nucleobase-protecting groups. The 2'-O-tom, 5'-O-(dimethoxytrityl)-protected ribonucleosides N2-acetyl-iso-cytidine, O2-(diphenyl-carbamoyl)-N6-isobutyryl-isoguanosine, N6-isobutyryl-N2-(methoxy-acetyl)purine-2,6-diamine ribonucleoside (=N8-isobutyryl-2-[(methoxyacetyl)amino]adenosine), 5-methyluridine, and 5,6-dihydro-uridine were prepd. by first introducing the nucleobase protecting groups and the dimethoxytrityl group, resp., followed by the 2'-O-tom group. The other presented 2'-O-tom, 5'-O-(dimethoxytrityl)-protected ribonucleosides inosine, 1-methyl-inosine 18, N6-iso-pent-2-enyl-adenosine, N6-methyl-adenosine, N6,N6-dimethyl-adenosine, 1-methyl-guanosine, N2-methylguanosine, N2,N2-dimethyl-guanosine, N6-(chloroacetyl)-1-methyladenosine, N6-(1S,2R)-2-[(tert-butyl)dimethyl-silyl]oxy-1-[2-(4-nitrophenyl)ethoxy]carbonylpropylamino carbonyl-adenosine (derived from L-threonine) and N4-acetyl-5-methyl-cytidine were prepd. by nucleobase transformation reactions from std., already 2'-O-tom-protected. Finally, all these nucleosides were transformed into the corresponding phosphoramidites, which are fully compatible with the assembly and deprotection conditions for std. RNA synthesis based on 2'-O-tom-protected monomeric building blocks. [on SciFinder (R)]

2005

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance

Johan Auwerx, Nouria Hernandez, Norman Lionel Moullan, Rajat Singh, Gilles Willemin

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Cold Spring Harbor Lab Press, Publications Dept2015

Preparation of aminoacylated tRNAs and analogues

EPFL2006