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

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

doi:10.1186/s12858-015-0043-8

Abstract

BackgroundThe 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.ResultsHere 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0043-8) contains supplementary material, which is available to authorized users.

Highlights

The 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system
Synthesis of 2-5As was performed with a human OAS1 isoform known to produce oligomers up to 30mers in vitro [5]
Expression and purification of OAS1 isoforms are straightforward compared with OAS2

Summary

Introduction

The 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. 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

Methods

Results

Discussion

Conclusion