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Signal-on/signal-off bead-based assays for the multiplexed monitoring of base excision repair activities by flow cytometry

Analytical and Bioanalytical Chemistry volume 414, pages 2029–2040 (2022)Cite this article

Abstract

As the support of all living kingdoms’ genetic information, the integrity of the DNA biomolecule must be preserved. To that goal, cells have evolved specific DNA repair pathways to thwart a large diversity of chemical substances and radiations that alter the DNA structure and lead to the development of pathologies such as cancers or neurodegenerative diseases. When dysregulated, activity rates of various actors of DNA repair can play a key role in carcinogenesis as well as in drugs resistance or hypersensitivity mechanisms. For the last 10 years, new complementary treatments have aimed at targeting specific enzymes responsible for such resistances. It is therefore crucial for biomedical research and clinical diagnosis to develop fast and sensitive tools able to measure the activity rate of DNA repair enzymes. In this work, a new assay for measuring enzymatic activities using microbeacons (µBs) is expounded. µB refers to microsphere functionalized by hairpin-shaped nucleic acid probes containing a single site-specific lesion in the stem and modified with chromophores. Following the processing of the lesion by the targeted protein, µB is cleaved and either lights off (signal-off strategy) or on (signal-on), depending on the use of fluorescent or profluorescent probes, respectively. After an optimization phase of the assay, we reported the combined analysis of restriction enzyme, AP-endonuclease, and DNA N-glycosylase by real-time monitoring followed by a flow cytometry measurement. As proofs of concept, we demonstrated the potential of the biosensor for highlighting DNA repair inhibitors and discriminating cell lines from their enzymatic activities.

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Acknowledgements

The authors would like to thank Yannick Rondelez for the careful proofreading of the manuscript.

Funding

This work was supported by the thesis funding program of CEA and the French National Research Agency (Labex ARCANE and CBH-EURGS, ANR-17-EURE-0003).

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Authors and Affiliations

  1. Laboratoire Gulliver, CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005, Paris, France

    Guillaume Gines

  2. University Grenoble Alpes, CEA, CNRS, IRIG, SyMMES-UMR5819, 38000, Grenoble, France

    Charlotte Brusa, Christine Saint-Pierre & Didier Gasparutto

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  1. Guillaume Gines
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  2. Charlotte Brusa
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DG acquired funds. DG and GG designed the study. GG, CB, and CSP performed experiments and analyzed the data. DG and GG wrote the manuscript.

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Correspondence to Guillaume Gines.

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Gines, G., Brusa, C., Saint-Pierre, C. et al. Signal-on/signal-off bead-based assays for the multiplexed monitoring of base excision repair activities by flow cytometry. Anal Bioanal Chem 414, 2029–2040 (2022). https://doi.org/10.1007/s00216-021-03849-9

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Keywords

  • Base excision repair
  • Bead-based assay
  • Flow cytometry
  • Real-time monitoring
  • Microbeacon