12th Carl Zeiss sponsored workshop on ... - Institut Fresnel

APPLICATIONS OF FLUORESCENCE CORRELATION SPECTROSCOPY
IN RNA BIOCHEMISTRY
Arne Werner *, Victor V. Skakun **, Petr V. Konarev ***, Patrick Ziegelmüller ****, Dmitri
I. Svergun *** and Ulrich Hahn ****
* Ludwigs-Maximilian University, Department of Chemistry, Institute for Physical Chemistry,
Munich, Germany
** Belarusian State University, Department of Systems Analysis, Minsk, Belarus
*** EMBL Outstation, c/o DESY, Hamburg, Germany and Institute of Crystallography,
Russian Academy of Sciences, Moscow, Russia
**** Hamburg University, Department of Chemistry, Institute for Biochemistry and
Molecular Biology, Hamburg, Germany
Arne.Werner@cup.uni-muenchen.de
An in vitro assay based on fluorescence correlation spectroscopy (FCS) is presented which
allows to investigate the kinetic behavior of human Dicer by different diffusion coefficients of
product and substrate. To investigate the linearity of the enzyme assay, FCS data of defined
solutions of two mobility species were analysed by different fitting routines, fitting
autocorrelation curves individually and globally. The optimised data analysis approach
allowed to monitor product formation with high accuracy.
As RNA does not emit fluorescence in the visible spectrum by itself, chemical or indirect
labeling is required to detect RNA by a fluorescence signal. The suitability of a
sulforhodamine B binding RNA aptamer to monitor RNA dynamics at the single molecule
level was tested. Additionally, molecular size and oligomerization state of the fluorophore-
RNA complexes could be characterized by FCS, FCCS and SAXS [1].
[1] Arne Werner, Petr V. Konarev, Dmitri I. Svergun and Ulrich Hahn, 'Characterisation of a
fluorophore binding RNA aptamer by Fluorescence Correlation Spectroscopy and Small
Angle X-Ray Scattering’, Analytical Biochemistry, 389, 52-62 (2009)