title: 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.
authors.affiliation: Department of Cancer Biology, Cleveland Clinic Foundation, Ohio 44195, USA.
authors.forename: R K
authors.initials: RK
authors.lastname: Maitra
authors.forename: G
authors.initials: G
authors.lastname: Li
authors.forename: W
authors.initials: W
authors.lastname: Xiao
authors.forename: B
authors.initials: B
authors.lastname: Dong
authors.forename: P F
authors.initials: PF
authors.lastname: Torrence
authors.forename: R H
authors.initials: RH
authors.lastname: Silverman
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D002678:Chimera; D017931:DNA Primers; D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis*; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D019892:eIF-2 Kinase
chemical_list: D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D016372:RNA, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease