Correlation of Selective Modifications to a 2‘,5‘-Oligoadenylate−3‘,5‘-Deoxyribonucleotide Antisense Chimera with Affinity for the Target Nucleic Acid and with Ability To Activate RNase L

The use of an antisense oligonucleotide to address a specific targeted RNA sequence and subsequent localized activation of the 2-5A-dependent RNase (RNase L) to effect selective RNA degradation is a new approach to the control of gene expression called 2-5A-antisense. The previously reported biological activity of the 2-5A:AS chimeric oligonucleotide [p5‘(A2‘p)3A-antiPKR1], directed against nucleotides 55−73 of the coding sequence of the PKR mRNA, has been used as a point of reference to examine the effect of introducing mismatches into the chimeric oligonucleotide, altering the chain length of the antisense domain of the chimeras, removal of the 5‘-monophosphate moiety, shortening the 2‘,5‘-oligoadenylate domain, and substitution of 3‘,5‘-linked 2‘-deoxyadenosine nucleotides for the 2-5A domain. The general formula for the novel chimeric oligonucleotides is p5‘(A2‘p)3A2‘p(CH2)4p(CH2)4p(5‘N3‘p) m N, where N is any nucleoside and m is any integer. When the biological activity of these new chimeric oligonucleotides was compared to that of the parent chimera, 2-5A-aPKR, for their ability to effect target PKR RNA cleavage in a cell-free and in an intact cell assay, it was determined that there was a close correlation between the activity of 2-5A-antisense chimeras and their affinity (T m) for a targeted nucleic acid. In addition, there was also a close correlation between activity of the 2-5A-antisense chimeras and their ability to activate the 2-5A-dependent RNase L.