Quantitative and real-time measurement of helicase-mediated intra-stranded G4 unfolding in bulk fluorescence stopped-flo
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Quantitative and real-time measurement of helicase-mediated intra-stranded G4 unfolding in bulk fluorescence stopped-flow assays Na-Nv Liu 1 & Lei Ji 1 & Qian Guo 1 & Yang-Xue Dai 1 & Wen-Qiang Wu 1 & Hai-Lei Guo 1 & Ke-Yu Lu 1 & Xiao-Mei Li 1 & Xu-Guang Xi 1,2 Received: 4 July 2020 / Revised: 3 August 2020 / Accepted: 10 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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 helicasemediated intramolecular G4 unfolding using stopped-flow assays but also offers a way to screen for inhibitors of G4 unfolding helicases as therapeutic drug targets. Keywords Enzymes . G-Quadruplexes . Kinetics . Fluorescence . Bioanalytical methods
Introduction G-quadruplexes (G4s) are special nucleic acid secondary structures formed by tandem repetitive G-rich DNA/RNA sequences [1–3]. In accordance with its preferential localization in functional genomic regions, G4 plays very important regulatory roles in telomere length maintenance, gene replication, transcription, and translation processes [3–5]. Refolding and Na-Nv Liu and Lei Ji contributed equally to this work as first authors. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02875-3) contains supplementary material, which is available to authorized users. * Xu-Guang Xi [email protected] 1
College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China
2
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
unfolding of G4 in the cell are spatially and temporally regulated by helicases [6–8]. Due to their globular conformation and intrinsic stability, helicases can unfold G4, but must recognize and unfold G4 through a mechanism different from the mechanism that processes duplex DNA. Compared with studies
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