A fluorescence correlation spectroscopy-based enzyme assay for human Dicer
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Arne Werner
Abstract
Here, we present an in vitro assay based on fluorescence correlation spectroscopy (FCS), which allows investigation of the kinetic behaviour of human Dicer. The assay is based on the different mobilities of substrate and product. The change of substrate mobility was independent of the choice of the fluorescence label, allowing exclusion of non-specific photophysical artefacts. Dicer and RNase III cleavage led to different product diffusion times. Single-stranded RNA did not change its mobility after cleavage by both double-strand-specific RNases. In agreement with the literature, the RNase activity of Dicer could be inhibited by substituting Ca2+ for Mg2+. In a defined system of two diffusion species of similar label and mobility differences, such as substrate and product, the linearity of the assay could be proven. An FCS-based enzyme assay is proposed, which allows monitoring of Dicer activity with high specificity in vitro.
©2012 by Walter de Gruyter Berlin Boston
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- Prelims
- Prelims
Artikel in diesem Heft
- Genes and Nucleic Acids
- The effect of cancer procoagulant on expression of metastatic and angiogenic markers in breast cancer and embryonic stem cell lines
- Characterization of RNA damage under oxidative stress in Escherichia coli
- Membranes, Lipids, Glycobiology
- Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells
- Cell Biology and Signaling
- Nucleocytoplasmic shuttling of human inositol phosphate multikinase is influenced by CK2 phosphorylation
- Apolipoprotein A5 internalized by human adipocytes modulates cellular triglyceride content
- Mammalian carboxylesterase (CES) releases GPI-anchored proteins from the cell surface upon lipid raft fluidization
- Proteolysis
- Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein
- Novel Techniques
- A fluorescence correlation spectroscopy-based enzyme assay for human Dicer
- Development of a host blood meal database: de novo sequencing of hemoglobin from nine small mammals using mass spectrometry
- Prelims
- Prelims
