A Convenient and Sensitive Fluorescence Resonance Energy Transfer Assay for RNase L and 2′,5′ Oligoadenylates

  • Chandar S. Thakur
  • Zan Xu
  • Zhengfu Wang
  • Zachary Novince
  • Robert H. Silverman
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 116)

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.

Key Words

Interferon RNase L FRET 2–5A 2′,5′ oligoadenylate endoribonuclease 

Notes

Acknowledgments

This work was supported by US Department of Defense Grant W81XWH-04-1-0055 (to R.H.S).

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Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Chandar S. Thakur
    • 1
    • 2
  • Zan Xu
    • 3
  • Zhengfu Wang
    • 2
  • Zachary Novince
    • 4
    • 2
  • Robert H. Silverman
    • 2
  1. 1.Department of ChemistryCleveland State UniversityCleveland
  2. 2.Department of Cancer BiologyCleveland Clinic FoundationCleveland
  3. 3.Ridgeway Biosystems Inc.Cleveland
  4. 4.Department of Biological, Geological, and Environmental SciencesCleveland State UniversityCleveland

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