title: Reverse Transcriptase in Action: FRET-Based Assay for Monitoring Flipping and Polymerase Activity in Real Time.
abstract: Reverse transcriptase (RT) of human immunodeficiency virus-1 (HIV-1) is a multifunctional enzyme that catalyzes the conversion of the single stranded viral RNA genome into double-stranded DNA, competent for host-cell integration. RT is endowed with RNA- and DNA-dependent DNA polymerase activity and DNA-directed RNA hydrolysis (RNase H activity). As a key enzyme of reverse transcription, RT is a key target of currently used highly active antiretroviral therapy (HAART), though RT inhibitors offer generally a poor resistance profile, urging new RT inhibitors to be developed. Using single molecule fluorescence approaches, it has been recently shown that RT binding orientation and dynamics on its substrate play a critical role in its activity. Currently, most in vitro RT activity assays, inherently end-point measurements, are based on the detection of reaction products by using radio-labeled or chemically modified nucleotides. Here, we propose a simple and continuous real-time Förster resonance energy transfer (FRET) based-assay for the direct measurement of RT's binding orientation and polymerase activity, with the use of conventional steady-state fluorescence spectroscopy. Under our working conditions, the change in binding orientation and the primer elongation step can be visualized separately on the basis of their opposite fluorescence changes and their different kinetics. The assay presented can easily discriminate non-nucleoside RT inhibitors from nucleoside RT inhibitors and determine reliably their potency. This one-step and one-pot assay constitutes an improved alternative to the currently used screening assays to disclose new anti-RT drugs and identify at the same time the class to which they belong.
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France.
authors.forename: K K
authors.initials: KK
authors.lastname: Sharma
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France.
authors.forename: F
authors.initials: F
authors.lastname: Przybilla
authors.affiliation: ‡Institute für Molekulare Medizin, Universitätsklinikum Schleswig-Holstein, Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Schleswig-Holstein, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Restle
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France.
authors.forename: C
authors.initials: C
authors.lastname: Boudier
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France.|§Département d'Information Médicale et de Biostatistiques, Hôpitaux Universitaires de Strasbourg, 1, pl de l'Hôpital, 67400 Strasbourg, France.
authors.forename: J
authors.initials: J
authors.lastname: Godet
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France.
authors.forename: Y
authors.initials: Y
authors.lastname: Mély
mesh_terms: D001681:Biological Assay / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D015497:HIV-1 / Q000201:enzymology*; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012194:RNA-Directed DNA Polymerase