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

Abstract

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.