Analogue d'acide nucléique | EPFL Graph Search

Les analogues d'acides nucléiques sont des composés qui sont structurellement similaires aux ARN et ADN présents dans la nature. Ils ont des utilisations pour la recherche en médecine et en biologie moléculaire. Les nucléotides, qui constituent les acides nucléiques, sont composées de trois parties : un squelette phosphate, un sucre pentose (ribose ou désoxyribose) et l'une des quatre bases nucléiques. Dans un analogue d'acide nucléique, n'importe laquelle de ces parties peut être modifiée. Cela résulte typiquement en une modification de l'appariement ou de l'empilement des bases (il existe ainsi des bases universelles pouvant s'apparier avec n'importe quelle autre base). Les analogues permettent aussi des modifications de la structure de la chaîne, en remplaçant le squelette phosphate. Un squelette de PNA permet par exemple d'obtenir une structure en triple hélice. Utilisations Analogue de nucléosides En médecine, plusieurs analogues de nucléosides sont employés comme

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

Rapid, Sensitive and Real-Time Multiplexing Platform for the Analysis of Protein and Nucleic-Acid Biomarkers

David Bernasconi, Nicolas Demierre, Lucienne Lagopoulos, Philippe Renaud, Raphaël Tornay

We describe a multiplexing technology, named Evalution, based on novel digitally encoded microparticles in microfluidic channels. Quantitative multiplexing is becoming increasingly important for research and routine clinical diagnostics, but fast, easy-to-use, flexible and highly reproducible technologies are needed to leverage the advantages of multiplexing. The presented technology has been tailored to ensure (i) short assay times and high reproducibility thanks to reaction-limited binding regime, (ii) dynamic control of assay conditions and real-time binding monitoring allowing optimization of multiple parameters within a single assay run, (iii) compatibility with various immunoassay formats such as coflowing the samples and detection antibodies simultaneously and hence simplifying workflows, (iv) analyte quantification based on initial binding rates leading to increased system dynamic range and (v) high sensitivity via enhanced fluorescence collection. These key features are demonstrated with assays for proteins and nucleic acids showing the versatility of this technology.

Amer Chemical Soc2015

Synthese und Paarungseigenschaften von pDNA und deren Anwendung bei der Entwicklung funktionalisierter Ribozyme

Damian Ackermann

Within this project methods for the preparation of the unnatural nucleic acids 3'-O-Methyl-pRNA and pDNA (3'-Desoxy-pRNA) were developed. The pairing properties of these two systems have been examined and compared with that of pRNA. All of the three systems do follow the same pairing rules. Pairing has been observed only on the Watson-Crick side forming duplexes in antiparallel direction. All three systems are autonomous pairing systems that do not interact with RNA or DNA. In this series pRNA forms the most stable duplexes. Nucleobase 9H-purin-2,6-diamin instead of adenin and 5-methyl-cytosine instead of cytosine were introduced to increase the stability of pDNA duplexes. A solution structure of the self-complementary pDNA sequence [MGAATTMG]2 was elucidated by NMR. It shows an antiparallel duplex twisted against the major groove with sligthly lefthanded helicity. In analogy to pRNA the interstrand stacking predominates with a inclination angel of approximately 45°. Proceeding form the NMR structure a MD simulation was performed, in which the duplex has strongly bent after a regeneration time of 1.5 ns; this bent shape was conserved during the remaining simulation time of 4.0 ns. Using automated solid phase synthesis pDNA/RNA hybrid sequences were prepared. Examining the pairing strength, the RNA-2'—>4'-pDNA linkage at the pDNA/RNA interface was observed to lead to more stable duplexes than the RNA-3'—>4'-pDNA linkage did. This kind of pDNA/RNA structures have been used to replace structure defining elements like hairpins or stem motifs by pDNA-duplexes in complex RNA structures. From the FMN binding RNA aptamer an analogous pDNA/RNA construct has been prepared, that was built of two hybrid sequences. The binding of FMN in the pDNA/RNA construct was shown by fluorescence spectroscopy and the dissociation constant K0 = 2.6·10-7 M was calculated. Using the same strategy a pDNA/RNA construct of the hammerhead ribozyme was prepared, having substrate cleavage activity in analogy to the natural ribozyme. Cleavage properties of the pDNA/RNA construct at different Mg2+ and Mn2+ concentrations and at different reaction temperatures showed the same trend as those from the natural ribozyme did. Under all measured conditions, the natural ribozyme was about 1.5 times as active as the pDNA/RNA construct. Additional functional groups were introduced at different positions of the pDNA/RNA hybrid sequences using a pre-functionalized 6'-bromopentyl allofuranosyl building bloc on which the bromide was substituted with MeNH2 resp. EtSH after solid phase synthesis. 25 different functionalized pDNA/RNA ribozyme constructs could be built up in a combinatorial way. From 17 out of the 25 constructs, the cleavage kinetics was measured and the cleavage rate evaluated. The substitution pattern of the most active pDNA/RNA ribozyme analogue was applied on the hammerhead RNA sequence and the doubly methylamino-substituted construct prepared. Compared to the natural hammerhead ribozyme, the cleavage rates of this construct were higher with a factor of 2.0 - 3.3 under all measured conditions.

EPFL2004

Preparation of aminoacylated tRNAs and analogues

EPFL2006

Synthese und Paarungseigenschaften von pDNA und deren Anwendung bei der Entwicklung funktionalisierter Ribozyme

Damian Ackermann

EPFL2004