title: In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis.
abstract: Liquid-phase electrophoresis either in the classical capillary format or miniaturized (chip CE) is a valuable tool for quality control of virus preparations and for targeting questions related to conformational changes of viruses during infection. We present an in vitro assay to follow the release of the RNA genome from a human rhinovirus (common cold virus) by using a molecular beacon (MB) and chip CE. The MB, a probe that becomes fluorescent upon hybridization to a complementary sequence, was designed to bind close to the 3' end of the viral genome. Addition of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known additive for reduction of bleaching and blinking of fluorophores in fluorescence microscopy, to the background electrolyte increased the sensitivity of our chip CE set-up. Hence, a fast, sensitive and straightforward method for the detection of viral RNA is introduced. Additionally, challenges of our assay will be discussed. In particular, we found that (i) desalting of virus preparations prior to analysis increased the recorded signal and (ii) the MB-RNA complex signal decreased with the time of virus storage at -70 °C. This suggests that 3'-proximal sequences of the viral RNA, if not the whole genome, underwent degradation during storage and/or freezing and thawing. In summary, we demonstrate, for two independent virus batches, that chip electrophoresis can be used to monitor MB hybridization to RNA released upon incubation of the native virus at 56 °C. Graphical Abstract Schematic of the study strategy: RNA released from HRV-A2 is detected by chip electrophoresis through the increase in fluorescence after genom complexation to a cognate molecular beacon.
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology (TU Wien), Getreidemarkt 9/164, 1060, Vienna, Austria.
authors.forename: Victor U
authors.initials: VU
authors.lastname: Weiss
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology (TU Wien), Getreidemarkt 9/164, 1060, Vienna, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Bliem
authors.affiliation: Department of Medical Biochemistry, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9, 1030, Vienna, Austria.
authors.forename: Irene
authors.initials: I
authors.lastname: Gösler
authors.affiliation: Department of Medical Biochemistry, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9, 1030, Vienna, Austria.
authors.forename: Sofiya
authors.initials: S
authors.lastname: Fedosyuk
authors.affiliation: Agilent Technologies, Hewlett-Packard-Straße 8, 76337, Waldbronn, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Kratzmeier
authors.affiliation: Department of Medical Biochemistry, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9, 1030, Vienna, Austria.
authors.forename: Dieter
authors.initials: D
authors.lastname: Blaas
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology (TU Wien), Getreidemarkt 9/164, 1060, Vienna, Austria. guenter.allmaier@tuwien.ac.at.
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
mesh_terms: D002839:Chromans / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D010850:Picornaviridae Infections / Q000821:virology*; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D012229:Rhinovirus / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D002839:Chromans; D005456:Fluorescent Dyes; D012367:RNA, Viral; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
keywords: Electrophoresis; Fluorescence; Molecular beacon; RNA uncoating; Virus