In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis
VerfasserWeiss, Victor U. ; Bliem, Christina ; Gösler, Irene ; Fedosyuk, Sofiya ; Kratzmeier, Martin ; Blaas, Dieter ; Allmaier, Günter
Erschienen in
Analytical and Bioanalytical Chemistry, Berlin ; Heidelberg, 2016, Jg. 408, H. 16, S. 4209-4217
ErschienenSpringer, 2016
Published version
DokumenttypAufsatz in einer Zeitschrift
Schlagwörter (EN)Virus / Electrophoresis / Molecular beacon / Fluorescence / RNA uncoating
URNurn:nbn:at:at-ubtuw:3-1618 Persistent Identifier (URN)
 Das Werk ist frei verfügbar
In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis [0.76 mb]
Supplementary material ESM 1 [0.77 mb]
Zusammenfassung (Englisch)

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 MBRNA 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.

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