A multiplex assay based on encoded microbeads conjugated to DNA NanoBeacons to monitor base excision repair activities by flow cytometry

• Published in: Biosensors & bioelectronics 2014-08, Vol.58, p.81-84
• Main Authors: Gines, Guillaume, Saint-Pierre, Christine, Gasparutto, Didier
• Format: Article
• Language: English
• Subjects: Base excision repair; Biological and medical sciences; Biosensing Techniques - instrumentation; Biosensors; Biotechnology; Chemical Sciences; DNA nanoprobes; DNA Probes - chemistry; DNA Probes - genetics; DNA Repair - genetics; Encoded microbeads; Equipment Design; Equipment Failure Analysis; Flow cytometry; Flow Cytometry - instrumentation; Fluorescence-based biosensor; Fundamental and applied biological sciences. Psychology; Life Sciences; Methods. Procedures. Technologies; Microarray Analysis - instrumentation; Miniaturization; Multiplex assay; Nanoparticles - chemistry; Nanoparticles - ultrastructure; Spectrometry, Fluorescence - instrumentation; Various methods and equipments
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2014.02.040

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. •Simple, easy, non-radioactive and electrophoresis-free DNA repair assay.•A fluorescent enzymatic assay using encoded microbeads coupled to a FACS platform.•Lesion-containing hairpin DNA nanoprobes are selective for repair enzymes.•The biosensor allows a multiplexed quantification of DNA repair activities.•A functional assay that works from purified enzymes or cell-free extracts.