title: Fluorescence resonance energy transfer analysis of RNase L-catalyzed oligonucleotide cleavage.
abstract: 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.
authors.affiliation: Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD 20892-0805, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Geselowitz
authors.forename: H
authors.initials: H
authors.lastname: Cramer
authors.forename: E M
authors.initials: EM
authors.lastname: Wondrak
authors.forename: M R
authors.initials: MR
authors.lastname: Player
authors.forename: P F
authors.initials: PF
authors.lastname: Torrence
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004722:Endoribonucleases / Q000737:chemistry*; D004789:Enzyme Activation; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D009843:Oligoribonucleotides; D012235:Rhodamines; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; D019793:Fluorescein