Abstract
Fluorescence correlation spectroscopy (FCS) allows determining diffusion and relaxation properties of fluorescent molecules. It requires only extremely small amounts of sample, down to picomolar concentrations, in an effective analysis volume of a few femtoliters. In essence, FCS determines the autocorrelation of fluorescence fluctuations caused by single labeled molecules passing through the confocal volume of a microscope equipped with a suitable detector; it permits investigating interactions of (macro)molecules, even in single cells. We present an FCS protocol for exploring, under in vitro conditions, the dynamic processes that take place during the early steps of virus infection. We cover two important issues of rhinovirus research, the kinetics of directional RNA release, and virus-receptor interactions exemplified by using human rhinovirus type A2 (HRV-A2) as a model.
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References
Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13:29–61
Rigler R, Widengren J (1990) Ultrasensitive detection of single molecules by fluorescence correlation spectroscopy. Bioscience 3:180–183
Haustein E (2007) Fluorescence correlation spectroscopy: novel variations of an established technique. Annu Rev Biophys Biomol Struct 36:151–169
Gosch M, Rigler R (2005) Fluorescence correlation spectroscopy of molecular motions and kinetics. Adv Drug Deliv Rev 57:169–190
Krieger J, Tódt K, Langowski J. “Biophysics of Macromolecules (B040): Practical Course Biophysics: Fluorescence Correlation Spectroscopy” http://www.dkfz.de/Macromol/teaching/files/fcs_practical.pdf
Krichevsky O, Bonnet G (2002) Fluorescence correlation spectroscopy: the technique and its applications. Rep Prog Phys 65:251–289
Harutyunyan S, Kumar M, Sedivy A et al (2013) Viral uncoating is directional: exit of the genomic RNA in a common cold virus starts with the poly-(A) tail at the 3′-end. PLoS Pathog 9:e1003270. doi:10.1371/journal.ppat.1003270
Wruss J, Rünzler D, Steiger C et al (2007) Attachment of VLDL receptors to an icosahedral virus along the 5-fold symmetry axis: Multiple binding modes evidenced by fluorescence correlation spectroscopy. Biochemistry 46:6331–6339
Duechler M, Skern T, Blaas D et al (1989) Human rhinovirus serotype 2: in vitro synthesis of an infectious RNA. Virology 168:159–161
Kremser L, Konecsni T, Blaas D et al (2004) Fluorescence labeling of human rhinovirus capsid and analysis by capillary electrophoresis. Anal Chem 76:4175–4181
Rye HS, Yue S, Wemmer DE et al (1992) Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res 20:2803–2812
Meseth UWT, Rigler R, Vogel H (1999) Resolution of fluorescence correlation measurements. Biophys J 76:1619–1631
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Harutyunyan, S., Sedivy, A., Köhler, G., Kowalski, H., Blaas, D. (2015). Application of FCS in Studies of Rhinovirus Receptor Binding and Uncoating. In: Jans, D., Ghildyal, R. (eds) Rhinoviruses. Methods in Molecular Biology, vol 1221. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1571-2_8
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DOI: https://doi.org/10.1007/978-1-4939-1571-2_8
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-1571-2
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