Dresden 2009 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 18: Regulation and Signaling

BP 18.5: Vortrag

Donnerstag, 26. März 2009, 11:00–11:15, HÜL 186

Influence of chemical modifications on siRNA strand separation and RISC target interaction studied by fluorescence cross-correlation spectroscopy in vivo — •Wolfgang Staroske, Thomas Ohrt, and Petra Schwille — Biophysics Group, BIOTEC, TU Dresden, Germany

Short double stranded RNA molecules have emerged as key regulators of gene expression in various organisms, both in the context of controlling developmental programs and as a defence mechanism to protect the genome against viruses and transposons. Short interfering 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 and a target RNA in vivo. We used a stable EGFP-Ago2 expressing 293 cell line, with endogenous expression levels suitable for FCS/FCCS measurements and designed a fluorescently labelled RNA, mimicking a target mRNA. By investigating the EGFP-Ago2 cell line and delivered fluorescently labelled siRNAs or targetRNA in vivo, we were able to gain new insights into siRNA strand separation, RISC loading and RISC target interactions. Our analysis of various chemical modified and fluorescently labelled siRNAs showed a correlation between chemical modification, passenger strand separation and gene silencing.