A Convenient and Sensitive Fluorescence Resonance Energy Transfer Assay for RNase L and 2′,5′ Oligoadenylates
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.
Key WordsInterferon RNase L FRET 2–5A 2′,5′ oligoadenylate endoribonuclease
This work was supported by US Department of Defense Grant W81XWH-04-1-0055 (to R.H.S).