Nouvelles sondes oligonucléotidiques fluorescentes ou paramagnétiques : applications à l'étude structurale des lésions de l'ADN et à leur réparation sur support.

DNA which is the support of genetic information is constantly subjected to damaging agents. These processes can lead to structural modifications of the DNA molecule and harmful biological consequences, such as mutagenesis or cancer. DNA damages can be repaired by enzymatic complexes which restore the original sequence of the biopolymer. In this study we were interested in structural aspects of DNA lesions and in their repair by the base excision repair (BER) and the reversion repair (RR) pathways. The present work consisted in developing a new tool in a biochip format aim at detecting these repairs activities by fluorescence measurement. Thus, hairpin-shaped damaged oligonucleotides were grafted on glass slides by a click chemistry approach. After optimization of the immobilization conditions, by click chemistry, this new biosensor was validated for the detection of repair activities using purified enzymes (glycosylases and AP-endonucleases) and cell extracts. Using a similar approach, we adapted this microarray to measure reversion repair and to screen DNA repair inhibitors. In a second part of this work, we applied the pulsed electron paramagnetic resonance (pulsed EPR) technique to study structural deformation induced by several DNA damages. To achieve this goal we developed a multiple site-specific labelling procedure of the DNA probes by nitroxide radicals. Then, pulsed EPR was used for the first time to detect an enzymatic DNA repair activity.L'ADN, support de l'information génétique, est constamment soumis à des stress l'endommageant. Ceci peut conduire à des modifications structurales de la molécule d'ADN et à des conséquences biologiques néfastes de type mutagénèse ou cancérogénèse. Les lésions de l'ADN peuvent être réparées par des complexes enzymatiques qui restaurent la séquence originale. Dans le présent travail nous nous sommes intéressés aux aspects structuraux des lésions de l'ADN et à leur réparation par excision de base (BER) ou par réversion (RR). Notre travail a consisté à développer un nouvel outil de type biopuce pour détecter ces activités de réparation par mesure de fluorescence. Pour cela des oligonucléotides lésés auto-complémentaires ont été immobilisés sur des lames de verre. Après avoir mis au point les conditions d'immobilisation, par la chimie click, nous avons validé ce nouveau biocapteur pour la détection d'activités de réparation d'enzymes purifiées (glycosylases et AP-endonucléases) ou au sein d'extraits cellulaires. Utilisant un principe similaire, nous avons adapté cette biopuce pour mesurer les activités de réparation par réversion ainsi que pour le screening d'inhibiteurs. Dans une seconde partie de ce travail, nous avons appliqué la technique de résonance paramagnétique électronique pulsée (RPE pulsée) pour étudier la déformation structurale induite par plusieurs dommages de l'ADN. Pour cela nous avons développé une méthode de multi-marquage de l'ADN par des radicaux nitroxydes. Cette technique a alors été appliquée pour la première fois à la détection d'une activité enzymatique de réparation de l'ADN

Nouvelles sondes oligonucléotidiques fluorescentes ou paramagnétiques : applications à l'étude structurale des lésions de l'ADN et à leur réparation sur support.

DNA which is the support of genetic information is constantly subjected to damaging agents. These processes can lead to structural modifications of the DNA molecule and harmful biological consequences, such as mutagenesis or cancer. DNA damages can be repaired by enzymatic complexes which restore the original sequence of the biopolymer. In this study we were interested in structural aspects of DNA lesions and in their repair by the base excision repair (BER) and the reversion repair (RR) pathways. The present work consisted in developing a new tool in a biochip format aim at detecting these repairs activities by fluorescence measurement. Thus, hairpin-shaped damaged oligonucleotides were grafted on glass slides by a click chemistry approach. After optimization of the immobilization conditions, by click chemistry, this new biosensor was validated for the detection of repair activities using purified enzymes (glycosylases and AP-endonucleases) and cell extracts. Using a similar approach, we adapted this microarray to measure reversion repair and to screen DNA repair inhibitors. In a second part of this work, we applied the pulsed electron paramagnetic resonance (pulsed EPR) technique to study structural deformation induced by several DNA damages. To achieve this goal we developed a multiple site-specific labelling procedure of the DNA probes by nitroxide radicals. Then, pulsed EPR was used for the first time to detect an enzymatic DNA repair activity.L'ADN, support de l'information génétique, est constamment soumis à des stress l'endommageant. Ceci peut conduire à des modifications structurales de la molécule d'ADN et à des conséquences biologiques néfastes de type mutagénèse ou cancérogénèse. Les lésions de l'ADN peuvent être réparées par des complexes enzymatiques qui restaurent la séquence originale. Dans le présent travail nous nous sommes intéressés aux aspects structuraux des lésions de l'ADN et à leur réparation par excision de base (BER) ou par réversion (RR). Notre travail a consisté à développer un nouvel outil de type biopuce pour détecter ces activités de réparation par mesure de fluorescence. Pour cela des oligonucléotides lésés auto-complémentaires ont été immobilisés sur des lames de verre. Après avoir mis au point les conditions d'immobilisation, par la chimie click, nous avons validé ce nouveau biocapteur pour la détection d'activités de réparation d'enzymes purifiées (glycosylases et AP-endonucléases) ou au sein d'extraits cellulaires. Utilisant un principe similaire, nous avons adapté cette biopuce pour mesurer les activités de réparation par réversion ainsi que pour le screening d'inhibiteurs. Dans une seconde partie de ce travail, nous avons appliqué la technique de résonance paramagnétique électronique pulsée (RPE pulsée) pour étudier la déformation structurale induite par plusieurs dommages de l'ADN. Pour cela nous avons développé une méthode de multi-marquage de l'ADN par des radicaux nitroxydes. Cette technique a alors été appliquée pour la première fois à la détection d'une activité enzymatique de réparation de l'ADN

Modeling and experimental validation of radiation-cellular media interactions in radiotherapy by photon activation of heavy elements

International audienceAn innovative approach using X-rays interactions with heavy elements seems to open a promising way of treatment for resistant cancers, such as high-grade gliomas. Such a technique is developed at the medical beam line of ESRF using monochromatic X-rays in the 50-100 keV range for the treatment of brain tumors [1,2]. The use of gold nanoparticles (GNP) to treat mice bearing subcutaneous tumors led to encouraging results [3]. However, the physical processes and biological impact of the photon activation of nanoparticles are not well understood. The experimental results cannot be explained from macroscopic dose calculations [4,5]. The aim of this study was to evaluate, at the cellular level, the dose enhancement in presence of nanoparticles and the properties of the secondary electrons production using the Monte Carlo transport code PENELOPE. The mean range, quantity and spectra of electrons produced from the interactions of X-rays with a GNP were calculated for 8 to 100 nm diameter GNP when irradiated with monochromatic photons from 30 keV to 2MeV. The dose enhancement was calculated in water at one micrometer around the GNP and in a cellular geometry considering homogeneous gold-water mixture. An increase of electron production by a factor 5 to 100 was observed in presence of GNP, for energies from 2 MeV to 30 keV respectively. The mean energy of the produced electrons increases with the beam energy, except after the K-edge of gold because of an enhancement of interactions with deeper atomic shells. An auto-absorption of low energy electrons for GNP with larger diameters was also observed. In the near future, we plan to calculate the dose in a cellular geometry considering multiple nanostructures

A DNA array based on clickable lesion-containing hairpin probes for multiplexed detection of base excision repair activities

International audienc

A universal and ready-to-use heterotrifunctional cross-linking reagent for facile synthetic access to sophisticated bioconjugates.

International audienceWe describe for the first time, the synthesis and some bioconjugation applications of an original heterotrifunctional cross-linking reagent (also named tripod) bearing three different bioorthogonal functional groups which are fully compatible amongst themselves. Contrary to the first generation tripod recently reported by us (Org. Biomol. Chem., 2008, 6, 3065), the use of an azido group instead of the nucleophile-sensitive active carbamate moiety enables us to reach the targeted chemical orthogonality without the use of temporary aminooxy- and thiol protecting groups. Thus, the preparation of sophisticated bioconjugates through the sequential derivatisation of the tripod by means of copper-mediated 1,3-dipolar cycloaddition, oxime ligation and aqueous compatible mild thiol-alkylation reactions, is significantly simpler and more convenient. The chemoselective bioconjugation protocols were optimised through the preparation of FRET cassettes based on cyanine and/or xanthene fluorescent dye pairs and subsequent anchoring to fragile biomolecules. The applicability of this universal cross-linking reagent was also illustrated by the preparation of biochips suitable for aflatoxin B1 detection through the SPIT-FRI method

Photo-activation therapy with high-Z nanoparticles: modelling at a micrometer level and experimental comparison

International audienceAn innovative approach using X-rays interactions with heavy elements seems to open a promising way of treatment for resistant cancers, such as high-grade gliomas. The use of gold nanoparticles to treat mice bearing subcutaneous tumors led to encouraging results [3]. However, the physical processes and biological impact of the photon activation of nanoparticles are not well understood. The aim of this study was to evaluate, at the cellular level, the dose enhancement in presence of nanoparticles and the properties of the secondary electrons production using the Monte Carlo transport code PENELOPE.At first, upstream studies were done around the behavior of gadolinium and gold nanoparticles (GdNP and AuNP) under irradiation. Then dose calculations were done in a cellular geometry containing gadolinium-water mixture in order to compare with experimentations realized on the ID17 beamline of ESRF

Grinding Lysis (GL): A microfluidic device for sample enrichment and mechanical lysis in one

International audienceRapid identification of health threatening bacteria and/or spores present in small concentration in sample fluids is of utmost importance. Efficient sample preparation and molecular detection aims to achieving this goal. Two processes must be conducted successively: the concentration of the targets in a small volume with simultaneous purification, followed by their lysis to provide accessible DNA templates. Conventional PCR is then used in situ to identify the targets. In this work we present an original approach combining an efficient concentration and purification of the bacteria and spores, a rapid and efficient grinding lysis step, working even for polluted samples, and the integration of the process in a semi-automated device. The method is very efficient and rapid: it can concentrate and detect less than 10 targets in 1 mL of sample, even if the sample is contaminated by some environmental contaminants. The most resistant spores are successfully lysed. In this study, we successively present the principle and performances of the method, and its integration on a in a semi-automated device. Perspectives to fully integrated system are discussed

A Triple Spin-Labeling Strategy Coupled with DEER Analysis to Detect DNA Modifications and Enzymatic Repair

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Analyse de l'ADN pendant une campagne de fouilles. Application à l'étude de coprolithes d'un site du Paléolithique supérieur (grotte Maldidier, Dordogne, France)

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Internalization of Iron Nanoparticles by Macrophages for the Improvement of Glioma Treatment

National audienceRationale: An alternative approach for the improvement of radiotherapy consists in increasing differentially the radiation dose between tumors and normal tissues using nanoparticles (NPs) that have been beforehand internalized into the tumor. These high-Z NPs can be photo-activated by monochromatic synchrotron X-rays, leading to a local dose enhancement delivered to the neighboring tumor cells. In order to carry the NPs into the tumor center, macrophages are currently under study for their phagocytosis and diapedesis abilities. In this study, we characterized J774A.1 macrophages’ internalization kinetics and subcellular distribution of two types of iron NPs.Materials and Methods: Three aspects of internalization were examined: first, the location of internalized NPs in J774A.1 macrophages following a 24h incubation with iron NPs was determined by optical microscopy after cell slicing. Subsequently, the iron intake after a 24h incubation with NPs was characterized using ICP-MS. The resulting cell viability was measured by Trypan Blue staining. Finally, the internalization dynamics were studied by absorbance measurements for 24 hours using a plate reader.Results: J774A.1 macrophages are able to endocytose NPs: we measured ~61±10 pg of internalized iron per macrophage (initial iron concentration: 0.3 mg/mL in culture medium. The cell survival was higher than 80% for all tested conditions (initial iron concentrations in culture medium between 0 and 2.4 mg/mL). Finally, we determined that the internalization kinetics for J774A.1 had a typical saturation time of one hour. These results are currently used in Monte Carlo simulations to model photoactivation processes.Conclusion: Macrophages seem to be promising vectors for NPs, being able to endocytose and retain them in their cytoplasm. Our following studies will attempt to shed light on their other potential abilities as “Trojan Horses”