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

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.