Ribonuclease L, a 2-5A-dependent enzyme: purification to homogeneity and assays for 2-5A binding and catalytic activity.
RNase L is a latent endonuclease found in reptiles, birds, and mammals. It is activated by the 2',5'-phosphodiester-linked oligoadenylates called 2-5A and has been implicated in the mechanism of action of interferon, as well as in a variety of other biological phenomena such as apoptosis. Covalent linkage of 2-5A to antisense oligonucleotides permits recruitment of RNase L for enhancement of antisense action. The purification of RNase L described herein and the assays for its detection and activation will help to provide further mechanistic details on how this unique nuclease functions and what its biochemical roles may be. In addition, such assays will facilitate the screening of 2-5A-antisense congeners for exploration of the potential therapeutic applications of RNase L.
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Laboratory of Medicinal Chemistry, Building 8, Room B2A02, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, 20892-0805, USA.
Wondrak
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Bayly
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Torrence
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D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002853:Chromatography, Liquid / Q000379:methods; D004722:Endoribonucleases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D051379:Mice; D019920:Photoaffinity Labels; D011072:Poly U / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000378:metabolism; D013379:Substrate Specificity
D019920:Photoaffinity Labels; D012335:RNA, Ribosomal; D011072:Poly U; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease