Enzyme assays for synthesis and degradation of 2-5As and other 2′-5′ oligonucleotides

Jesper Buchhave Poulsen, Karina Hansen Kjær, Just Justesen, Pia Møller Martensen

The 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7. Here we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2'-5' oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD(+) as substrates is also presented. The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2'-5' oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays. This paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.