NDLI: In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis

Content Provider	Springer Nature Link
Author	Weiss, Victor U. Bliem, Christina Gösler, Irene Fedosyuk, Sofiya Kratzmeier, Martin Blaas, Dieter Allmaier, Günter
Copyright Year	2016
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
Starting Page	4209
Ending Page	4217
Page Count	9
File Format	PDF
ISSN	16182642
Journal	Analytical and Bioanalytical Chemistry
Volume Number	408
Issue Number	16
e-ISSN	16182650
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2016-03-28
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Virus Electrophoresis Molecular beacon Fluorescence RNA uncoating Analytical Chemistry Biochemistry Laboratory Medicine Characterization and Evaluation of Materials Food Science Environmental Monitoring/Analysis
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry Biochemistry

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