Berlin 2008 – scientific programme

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BP: Fachverband Biologische Physik

BP 20: Regulation and Signaling

BP 20.4: Talk

Thursday, February 28, 2008, 10:45–11:00, C 243

Fluorescence cross-correlation spectroscopy measurements in vivo reveal the asymmetric incorporation of siRNAs into RISC and the localisation of the complex in human cells — Thomas Ohrt, •Wolfgang Staroske, Jörg Mütze, and Petra Schwille — Biophysics Group, BIOTEC/TU Dresden

Short double stranded RNA molecules have emerged as key regulators of gene expression, controlling developmental programs as well as functioning as a defence mechanism against viruses and transposons. Small RNAs use Argonaute-containing complexes called RNA-Induced Silencing Complex (RISC) to identify cognate RNA transcripts whose expression is to be silenced. By combining laser scanning microscopy, fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) and biochemical methods, we have exploited the interaction of short interfering RNAs with RISC in vivo. We established a functional and stable EGFP-Ago2 expressing 293 cell line, with expression levels suitable for FCS/FCCS. Using this in vivo system combined with highly sensitive FCS and FCCS it is possible to gain vast information on relative binding, concentration and mobility. Analysis of various microinjected fluorescently labelled siRNAs with FCCS showed the asymmetry dependent incorporation of the antisense strand into RISC over time in human cells. Measurements in various cell compartments showed the localisation of loaded RISC complex in human cells.