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Quantitative and real-time measurement of helicase-mediated intra-stranded G4 unfolding in bulk fluorescence stopped-flow assays.

Authors
  • Liu, Na-Nv1
  • Ji, Lei1
  • Guo, Qian1
  • Dai, Yang-Xue1
  • Wu, Wen-Qiang1
  • Guo, Hai-Lei1
  • Lu, Ke-Yu1
  • Li, Xiao-Mei1
  • Xi, Xu-Guang2, 3
  • 1 College of Life Sciences, Northwest A&F University, Yangling, 712100, Shaanxi, China. , (China)
  • 2 College of Life Sciences, Northwest A&F University, Yangling, 712100, Shaanxi, China. [email protected] , (China)
  • 3 Laboratoire de Biologie et Pharmacologie Appliquée, Ecole Normale Supérieure de Cachan, Centre National de la Recherche Scientifique, Université Paris-Saclay, 61 Avenue du Président Wilson, 94235, Cachan, France. [email protected] , (France)
Type
Published Article
Journal
Analytical and Bioanalytical Chemistry
Publisher
Springer-Verlag
Publication Date
Nov 01, 2020
Volume
412
Issue
27
Pages
7395–7404
Identifiers
DOI: 10.1007/s00216-020-02875-3
PMID: 32851458
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

G-Quadruplexes (G4s) are thermodynamically stable, compact, and poorly hydrated structures that pose a potent obstacle for chromosome replication and gene expression, and requiring resolution by helicases in a cell. Bulk stopped-flow fluorescence assays have provided many mechanistic insights into helicase-mediated duplex DNA unwinding. However, to date, detailed studies on intramolecular G-quadruplexes similar or comparable with those used for studying duplex DNA are still lacking. Here, we describe a method for the direct and quantitative measurement of helicase-mediated intramolecular G-quadruplex unfolding in real time. We designed a series of site-specific fluorescently double-labeled intramolecular G4s and screened appropriate substrates to characterize the helicase-mediated G4 unfolding. With the developed method, we determined, for the first time to our best knowledge, the unfolding and refolding constant of G4 (≈ 5 s-1), and other relative parameters under single-turnover experimental conditions in the presence of G4 traps. Our approach not only provides a new paradigm for characterizing helicase-mediated intramolecular G4 unfolding using stopped-flow assays but also offers a way to screen for inhibitors of G4 unfolding helicases as therapeutic drug targets. Graphical abstract.

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