Selective RNA cleavage by isolated RNase L activated with 2-5A antisense chimeric oligonucleotides.

Publisher Summary Cellular ribonucleases often play essential roles in the mechanism of action of antisense oligonucleotides (ODNs) because of their ability to destroy target RNA molecules. RNase L is present in a wide range of cell types in higher vertebrate organisms in either an inactive form or as a highly potent, single-strand-specific endoribonuclease. Activation of RNase L occurs in response to the binding of short, 2',5'-linked oligoadenylates collectively referred to as 2-5A. The 2-5A system is part of the antiviral mode of action of interferons. Interferon treatment of mammalian cells results in the induction of a family of 2-5A synthetases that require double-stranded RNA (dsRNA) to catalyze the production of 2-5A from ATP. Virus-infected cells produce and secrete type I interferons that bind to receptors on cells, leading to enhanced levels of 2-5A synthetases and RNase L. Thereafter, viral dsRNA stimulates the production of 2-5A, resulting in RNase L activity and suppression of the viral infection. The ability of RNase L to be activated by 2-5A has been exploited for the purpose of degrading RNA molecules of choice. 2-5A is modified by attachment through linkers to an antisense cassette. The resulting 2-5A antisense species binds to both an RNA target and to RNase L. Activation of RNase L by the 2-5A moiety causes targeted degradation of the bound RNA molecule. Therefore, 2-5A antisense is a derivative of 2-5A that forces RNase L to selectively cleave RNA targets. The selectivity is because of a proximity effect from directing the activated RNase L to the RNA target. Furthermore, addition of the linker/antisense domains to 2-5A suppresses general 2-5A activity. This chapter describes methods for performing targeted degradation of RNA with purified RNase.