High yield synthesis, purification and characterisation of the RNase L activators 5'-triphosphate 2'-5'-oligoadenylates

Abstract

Upon viral infection, double-stranded viral RNA is detected very early in the host cell by several cellular 2'-5' oligoadenylate synthetases, which synthesize 2'-5' adenylate oligonucleotides that activate the cellular RNase L, firing an early primary antiviral response through self and non-self RNA cleavage. Transfecting cells with synthetic 2'-5' adenylate oligonucleotides activate RNase L, and thus provide a useful shortcut to study the early steps of cellular and viral commitments into this pathway. Defined 2'-5' adenylate oligonucleotides can be produced in vitro, but their controlled synthesis, purification, and characterisation have not been reported in detail. Here, we report a method suitable to produce large amounts of 2-5As of defined lengths in vitro using porcine OAS1 (pOAS) and human OAS2 (hOAS). We have synthesized a broad spectrum of 2-5As at the milligram scale and report an HPLC-purification and characterisation protocol with quantified yield for 2-5A of various lengths.