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.
Thakur
Chandar S
CS
Department of Chemistry, Cleveland State University, OH, USA.
Xu
Zan
Z
Wang
Zhengfu
Z
Novince
Zachary
Z
Silverman
Robert H
RH
D015088:2',5'-Oligoadenylate Synthetase / Q000235:genetics / Q000378:metabolism; D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D012680:Sensitivity and Specificity
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; C023505:2',5'-oligoadenylate; D012313:RNA; D015088:2',5'-Oligoadenylate Synthetase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease