Book of Abstracts: Albany 2005
Small Interfering (si)RNA Degradation in Cell Extracts: Real-Time Monitoring by Ensemble and Single Molecule Fluorescence Resonance Energy Transfer (FRET)
Short noncoding RNAs are increasingly recognized as key regulators of essential cellular processes such as RNA interference. A better understanding of the processes by which such RNAs are degraded is necessary to expand our knowledge of these processes and our ability to harness them. To this end we have developed novel fluorescence resonance energy transfer (FRET) assays to monitor in real-time the degradation kinetics of short RNAs by a purified RNase and S100 cytosolic HeLa cell extract. An unstructured single-strand of RNA is found to be degraded more rapidly than a corresponding stem-loop RNA under all conditions tested except for low concentrations of cell extract, showing that secondary structure confers protection against RNase activity. The assay also allows for the quantitative comparison of inhibitors such as Contrad70® and aurin tricarboxylic acid (ATA). In addition, gel electrophoretic FRET analysis confirms that HeLa cell extract is dominated by 5' to 3' exonucleolytic activity (J. Am. Chem. Soc. 125, 14230-14231 (2003)).
We now have turned our attention to probing the stability and protein binding properties of small interfering (si)RNAs by similar FRET assays. Although the chemically synthesized mimics of cellular siRNAs shape up to become a multi-billion dollar asset in gene therapy and functional genomics, relatively little is known to date about their cellular fate. We are working on fundamentally changing that by using our FRET assays, both in bulk and at the single molecule level, to monitor in real-time the degradation and unwinding of siRNAs in human cell extracts. We find that the single-stranded antisense RNA is rapidly degraded by the purified, G-specific RNase T1, while hybridization with the complementary RNA or DNA strongly protects it, as expected. By contrast, a corresponding RNA-DNA hybrid is by far most rapidly degraded in cytosolic HeLa cell extract, while the single-stranded antisense strand and its RNA-RNA hybrid are increasingly protected. This initially unexpected behavior may relate to the fact that double-stranded siRNAs, and subsequently their single-stranded antisense strands in complex with RISC, mediate RNA interference and thus are protected by protein binding inside a human cell, while an RNA-DNA hybrid may be subject to a cellular RNase H activity. Finally, to better understand the mechanism by which the 19-kDa protein p19 from the plant pathogenic Carnation Italian ringspot virus (CIRV) sequesters anti-viral siRNAs, we are utilizing FRET assays to measure binding and dissociation rate constants of p19-siRNA complexes and thus determine true equilibrium binding affinities. If chosen for oral presentation, the newest results of these lines of experimentation will be described.
Sarah A. Uhler
Dow Corning Assistant Professor of Chemistry