title: A multiplex assay based on encoded microbeads conjugated to DNA NanoBeacons to monitor base excision repair activities by flow cytometry.
abstract: We reported here a new assay to detect base excision repair activities from purified enzymes, as well as in cell-free extracts. The multiplex format rests upon the encoding of magnetic beads with the fluorophore Alexa 488, thanks to a fast and original procedure. Fluorescently encoded microbeads are subsequently functionalized by lesion-containing DNA NanoBeacons labeled with the fluorophore/quencher pair Cyanine 5/BHQ2. Probes cleavage, induced by targeted enzymes leads to Cyanine 5 signal enhancement, which is finally quantified by flow cytometry. The multiplex assay was applied to the detection of restriction enzymes activities as well as base excision repair processes. Each test requires only 500fmol of DNA substrate, which constitutes great sensitivity compared to other BER functional assays. The present biosensor is able to detect both uracil DNA N-glycosylase (UNG) and AP-endonuclease 1 (APE1) within few nanograms of nuclear extract. Additionally, we demonstrated that the corresponding assay has potential application in DNA repair inhibitor search. Finally, the current multiplexed tool shows several advantages in comparison to other functional BER assays with no need of electrophoretic separation, straightforward, easy and reproducible functionalization of encoded microbeads and a high stability of DNA probes in cell-free extracts.
authors.affiliation: Laboratoire des Lésions des Acides Nucléiques, INAC/SCIB UMR_E3 CEA/UJF-Grenoble 1/CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Gines
authors.affiliation: Laboratoire des Lésions des Acides Nucléiques, INAC/SCIB UMR_E3 CEA/UJF-Grenoble 1/CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Saint-Pierre
authors.affiliation: Laboratoire des Lésions des Acides Nucléiques, INAC/SCIB UMR_E3 CEA/UJF-Grenoble 1/CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France. Electronic address: didier.gasparutto@cea.fr.
authors.forename: Didier
authors.initials: D
authors.lastname: Gasparutto
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation*; D046228:Microarray Analysis / Q000295:instrumentation*; D008904:Miniaturization; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence / Q000295:instrumentation
chemical_list: D015342:DNA Probes
keywords: Base excision repair; DNA nanoprobes; Encoded microbeads; Flow cytometry; Fluorescence-based biosensor; Multiplex assay