A fluorescence correlation spectroscopy-based enzyme assay for human Dicer.
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.
Institute for Biochemistry and Molecular Biology, Department of Chemistry, MIN-Faculty, Hamburg University, Martin-Luther-King Platz 6, D-20146 Hamburg, Germany.
Arne
A
Werner
Victor V
VV
Skakun
Patrick
P
Ziegelmüller
Ulrich
U
Hahn
D002118:Calcium / Q000378:metabolism; D002232:Carbocyanines / Q000032:analysis; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D002232:Carbocyanines; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C085321:cyanine dye 5; D012313:RNA; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases; D008274:Magnesium; D002118:Calcium