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.
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 46:6331–6339PubMedCrossRefGoogle Scholar
Duechler M, Skern T, Blaas D et al (1989) Human rhinovirus serotype 2: in vitro synthesis of an infectious RNA. Virology 168:159–161PubMedCrossRefGoogle Scholar
Kremser L, Konecsni T, Blaas D et al (2004) Fluorescence labeling of human rhinovirus capsid and analysis by capillary electrophoresis. Anal Chem 76:4175–4181PubMedCrossRefGoogle Scholar
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 20:2803–2812PubMedCrossRefPubMedCentralGoogle Scholar