Catalytic Cleavage of an RNA Target by 2–5A Antisense and RNase L

Abstract

2-5A antisense (2-5A-AS) molecules are chimeric oligonucleotides that cause 2-5A-dependent RNase (RNase L) to catalyze the selective cleavage of RNA in human cells. These composite nucleic acids consist of a 5'-monophosphorylated, 2',5'-linked oligoadenylate known as 2-5A (an activator of RNase L) covalently attached to antisense 3',5'-oligodeoxyribonucleotides. Here, we characterize the targeted cleavage of the double-stranded RNA-dependent protein kinase (PKR) mRNA by purified, recombinant human RNase L. A 2-5A-AS chimera, which contains complementary sequence to PKR mRNA, and unmodified 2-5A, which causes general RNA decay, were about 20- and 40-fold more active, respectively, than 2-5A-AS chimeras in which the DNA domains are not complementary to sequences in PKR mRNA. Directed cleavage was efficient because each 2-5A-AS chimera targeted many RNA molecules. Moreover, RNase L caused the catalytic cleavage of the RNA target (kcat of approximately 7 s-1). The precise sites of PKR mRNA cleavage caused by 2-5A-AS were mapped, using a primer extension assay, to phosphodiester bonds adjacent to the 3' terminus of the chimera binding site (5' on the RNA target) as well as within the chimera's oligonucleotide binding site itself. The selectivity of this approach is shown to be provided by the antisense arm of the chimera, which places the RNA target in close proximity to the RNase.

Targeting RNA decay with 2′,5′ oligoadenylate-antisense in respiratory syncytial virus-infected cells

“…Our findings support previous reports that covalent linkage of 2-5A to an antisense oligonucleotide enhances rates of decay for the target RNAs in cells through the action of RNase L (12,23,24). In particular, the data show that spA 4 -antiRSV3Ј-3ЈT͞(8281-8299) attracts RNase L to complementary sequences in RSV M2 mRNA, causing its degradation.…”

“…The 2-5A-chimeras-e.g. as spA 4 -antiRSV3Ј-3ЈT͞(8281-8299)-did not cause the general decay of RNA, probably because they are relatively inefficient activators of RNase L (23). Nevertheless, when target RNAs were brought into close proximity to RNase L, the enzymatic activity was sufficient to cause the rapid cleavage of the RNA target.…”

“…In the 2-5A-antisense approach, RNase L is converted to a highly specific endoribonuclease capable of selectively cleaving individual RNA targets. This previously was demonstrated in cell-free systems against a modified HIV-1 vif mRNA (12) and against an mRNA target encoding the double-stranded RNA-activatable protein kinase, PKR (23). When applied to HeLa cells, the 2-5A-antisense resulted in the ablation of PKR mRNA and enzymatic activity (24).…”

The Quest for an Efficacious Antiviral for Respiratory Syncytial Virus

“…For example , in a Daudi cell-free extract, chimera Iva (Table 1) brought about cleavage of RNA construct target TAR:A25:vif; however, chimera IVb, bearing the inefficient 5′-non-phosphorylated RNase L activator, A2′p5′A′2p5′A′2p5′A, was unable to produce TAR:A25:vif cleavage. Similarly, chimera Vb, directed against the PKR mRNA and bearing the same 5′-nonphosphorylated tetrameric 2′,5′-oligoadenylate, could not effect either cell-free or intact cell PKR RNA cleavage under conditions where chimera Va was highly effective at cutting PKR mRNA (Maitra et al, 1995;Maran et al, 1994). A similar result was obtained with chimera Ia, one of the first 2-5A-antisense formulations that exhibited anti-RSV activity (Cirino et al, 1997).…”

Targeted therapy of human malignant glioma in a mouse model by 2-5A antisense directed against telomerase RNA

“…2-5A antisense refers to a class of oligonucleotide therapeutic agents that cause the catalytic breakdown of target RNA molecules Maran et al, 1994;Maitra et al, 1995;Cirino et al, 1997). These drugs contain a 2',5'-linked tetraadenylate (2-5A) covalently attached through linkers to antisense oligonucleotides.…”