Abstract
Interferon action against viruses is mediated in part through a ribonucleic acid (RNA) decay pathway known as the 2–5A system. Unusual 5′-triphosphorylated, 2′,5′-linked oligoadenylates (2–5A) are produced in mammalian cells by interferon-inducible 2–5A synthetases (OAS) in response to viral double-stranded RNA. 2–5A activates a uniquely regulated endoribonuclease, RNase L, resulting in the cleavage of single-stranded viral and cellular RNAs, thus suppressing viral replication. In addition, RNase L was recently identified as a strong candidate for the hereditary prostate cancer 1 susceptibility allele. RNase L is ubiquitously expressed at basal levels in a wide range of mammalian cell types. Conventional RNase L assays, which can be inconvenient and cumbersome, typically involve cleavage of radioactively labeled RNA species or of endogenous ribosomal RNA. Here we describe a convenient, rapid, nonradioactive, and relatively inexpensive fluorescence resonance energy transfer (FRET) that may be used to accurately measure levels of either 2–5A or RNase L activity with a high degree of specificity and sensitivity. The RNA probe used in the FRET assay was designed based on a region of respiratory syncytial genomic RNA. We demonstrate the utility of our FRET assay with several novel biostable analogs of 2–5A.
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Acknowledgments
This work was supported by US Department of Defense Grant W81XWH-04-1-0055 (to R.H.S).
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Thakur, C.S., Xu, Z., Wang, Z., Novince, Z., Silverman, R.H. (2005). A Convenient and Sensitive Fluorescence Resonance Energy Transfer Assay for RNase L and 2′,5′ Oligoadenylates. In: Carr, D.J.J. (eds) Interferon Methods and Protocols. Methods in Molecular Medicine™, vol 116. Humana Press. https://doi.org/10.1385/1-59259-939-7:103
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DOI: https://doi.org/10.1385/1-59259-939-7:103
Publisher Name: Humana Press
Print ISBN: 978-1-58829-418-0
Online ISBN: 978-1-59259-939-4
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