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Fluorescence resonance energy transfer analysis of RNase L-catalyzed oligonucleotide cleavage. 2000
Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD 20892-0805, USA.
A method is described for monitoring the cleavage of an oligoribonucleotide substrate by the 2-5A-dependent RNase L based on fluorescence resonance energy transfer (FRET). The oligoribonucleotide, rC11U2C7, was labeled covalently at its 5'-terminus with fluorescein and at its 3'-terminus with rhodamine to provide a substrate for RNase L. On cleavage, the fluorescence at 538 nm (with 485 nm excitation) increased by a factor of 2.8, allowing real-time quantitation of the reaction progress. The method was performed easily in a 96-well plate format and allowed quantitative high throughput analyses of RNase L activity with different activators.
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