An in silico and in vitro pipeline for the rapid screening of helicase modulators

Authors

  • Eleni Papakonstantinou 1Laboratory of Genetics, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, Athens 2Lab of Molecular Endocrinology, Center of Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens
  • Flora Bacopoulou Center for Adolescent Medicine and UNESCO Chair on Adolescent Health Care, First Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, Aghia Sophia Children’s Hospital, Athens
  • Vasileios Megalooikonomou Computer Engineering and Informatics Department, School of Engineering, University of Patras, Patras
  • Aspasia Efthimiadou Hellenic Agricultural Organization-Demeter, Institute of Soil and Water Resources, Department of Soil Science of Athens, Lycovrisi
  • Dimitrios Vlachakis 1Laboratory of Genetics, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens 2Lab of Molecular Endocrinology, Center of Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens 6Department of Informatics, Faculty of Natural and Mathematical Sciences, King’s College London, Strand, London, UK

DOI:

https://doi.org/10.14806/ej.25.0.927

Keywords:

helicase modulator, enzyme activity, luminescence, screening

Abstract

To evaluate the potency of potential helicase modulators, we developed an assay of helicase enzyme activity. Using a DNA or RNA biotin labelled oligonucleotide and after the addition of a recombinant helicase, the nucleic acid unwinds, causing the emission of luminescence, which is quantified with a particular antibody. In our assay, one of the DNA oligos was biotinylated, while the other was labelled with digoxygenin (DIG), both in their 5’ termini. The biotin molecule immobilises the DNA duplex on a neutravidin-coated plate and the helicase activity is measured through the unwinding of DNA, due to ATP activation. The subsequent release of DIG-labelled oligos results in a luminescence signal measured with a chemiluminescence antibody. Our goal was to provide a high throughput screening method for potential helicase inhibitors. The method described in this paper has been demonstrated to be fast, easy and reproducible and doesn’t use radiochemicals.

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Published

2020-02-14

Issue

Vol. 25

Section

Research Papers

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