Développement de nouveaux outils analytiques à base d'acides nucléiques aptamères pour la détection de petites molécules

La détection de petites molécules est d'un grand intérêt dans les domaines pharmaceutique, environnemental, alimentaire et de la biologie clinique. Les aptamères, sélectionnés par la méthode SELEX (pour Systematic Evolution of Ligands by Exponential Enrichment), sont des oligonucléotides qui se lient à une cible donnée avec une affinité et une spécificité importantes. L'objectif de ce travail est d'établir de nouvelles méthodologies analytiques basées sur l'utilisation des aptamères pour la détection de petites molécules. Dans un premier temps, une méthodologie par électrophorèse capillaire, dérivée du concept de déplacement du brin complémentaire de l'aptamère, est décrite pour la détection simultanée de plusieurs analytes dans un seul capillaire. La deuxième étude se focalise sur le développement d'un aptacapteur colorimétrique simple, rapide et peu coûteux, qui utilise le concept général de protection enzymatique de l'aptamère et les nanoparticules d'or en tant que système de transduction. Enfin, deux méthodes par polarisation de fluorescence, basées sur le concept de déplacement (du brin complémentaire ou de l'aptamère lui-même), sont présentées afin d'accroitre les potentialités des aptacapteurs dédiés à la détection des petites molécules.Small biomolecule detection is of great interest and importance in the pharmaceutical, environmental, food and clinical fields. Aptamers, selected by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), are oligonucleotides that bind to a target with high affinity and specificity. The objective of the work is to establish novel methodologies of aptamer-based assays for the small biomolecule detection. In the first work, a rationalized capillary electrophoresis strategy, derived from the structure-switching aptamer concept, is described for the design of simultaneous detection of multiple analytes. The second work based on a gold nanoparticle colorimetric sensing strategy allows a rapid, label-free, homogeneous assay for small molecule using an aptamer enzymatic cleavage protection strategy. In the third work, two aptamer-based fluorescence polarization approaches, using the displacement concept, are described to improve the potentialities of the small molecule-dedicated aptasensors.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau Proteins

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Ultrafast capillary electrophoresis isolation of DNA aptamer for the PCR amplification-based small analyte sensing

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Aptamer-based enantioselective competitive binding assay for the trace enantiomer detection.

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Noncompetitive Fluorescence Polarization Aptamer-based Assay for Small Molecule Detection

International audienceIn this paper, a new fluorescence polarization (FP) assay strategy is described reporting the first demonstration of a noncompetitive FP technique dedicated to the small molecule sensing. This approach was based on the unique induced-fit binding mechanism of nucleic acid aptamers which was exploited to convert the small target binding event into a detectable fluorescence anisotropy signal. An anti-l-tyrosinamide DNA aptamer, labeled by a single fluorescent dye at its extremity, was employed as a model functional nucleic acid probe. The DNA conformational change generated by the l-tyrosinamide binding was able to induce a significant increase in the fluorescence anisotropy signal. The method allowed enantioselective sensing of tyrosinamide and analysis in practical samples. The methodology was also applied to the l-argininamide detection, suggesting the potential generalizability of the direct FP-based strategy. Such aptamer-based assay appeared to be a sensitive analytical system of remarkable simplicity and ease of use

Covalently bonded DNA aptamer chiral stationary phase for the chromatographic resolution of adenosine.

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Chiral Resolution Capabilities of DNA Oligonucleotides

Herein, we studied the chiral resolution properties of a repertoire of arbitrarily chosen DNA oligonucleotides (ON). Ten oligonucleotidic sequences characterized by diverse base compositions, sizes, and structural features, ranging from secondary structure-free homo-oligonucleotides to duplex, hairpin, and three-way junction architectures, were investigated as potential chiral selectors. Their enantioselective features were assessed by using ONs as running buffer additives in partial-filling capillary electrophoresis. It was shown that all the screened sequences displayed enantiodiscrimination capabilities toward small aromatic compounds. Under (sub)­millimolar DNA concentration conditions, the combination of only three oligonucleotidic sequences provided the chiral resolution of around 20 racemates, including drugs, illegal drugs, amino-acids, and nucleosides. This work represents the first demonstration of such analyte selectivity spectrum for nucleic acid-based chiral separation tools

ATP7B-Deficient Hepatocytes Reveal the Importance of Protein Misfolding Induced at Low Copper Concentration

International audienceCopper is a transition metal essential for human life. Its homeostasis is regulated in the liver, which delivers copper to the whole body and excretes its excess outside the organism in the feces through the bile. These functions are regulated within hepatocytes, and the ATP7B copper transporter is central to making the switch between copper use and excretion. In Wilson disease, the gene coding for ATP7B is mutated, leading to copper overload, firstly, in the liver and the brain. To better understand the role of ATP7B in hepatocytes and to provide a smart tool for the development of novel therapies against Wilson disease, we used the CrispR/Cas9 tool to generate hepatocyte cell lines with the abolished expression of ATP7B. These cell lines revealed that ATP7B plays a major role at low copper concentrations starting in the micromolar range. Moreover, metal stress markers are induced at lower copper concentrations compared to parental cells, while redox stress remains not activated. As shown recently, the main drawback induced by copper exposure is protein unfolding that is drastically exacerbated in ATP7B-deficient cells. Our data enabled us to propose that the zinc finger domain of DNAJ-A1 would serve as a sensor of Cu stress. Therefore, these Wilson-like hepatocytes are of high interest to explore in more detail the role of ATP7B