Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides

Antisense oligonucleotides (ASOs) are synthetic oligonucleotides that alter expression of disease-associated transcripts via Watson–Crick hybridiza-tion. ASOs that function through RNase H or the RNA-induced silencing complex (RISC) result in en-zymatic degradation of target RNA. ASOs designed to sterically block access of proteins to the RNA mod-ulate mRNA metabolism but do not typically cause degradation. Here, we rationally design steric block-ing ASOs to promote mRNA reduction and character-ize the terminating mechanism. Transfection of ASOs complementary to constitutive exons in STAT3 and Sod1 results in greater than 70 % reduction of mRNA and protein. The ASOs promote aberrant exon skip-ping and generation of premature termination codon (PTC)-containing mRNAs. We inhibit the nonsense-mediated mRNA decay (NMD) pathway and show that the PTC-containing mRNAs are recognized by the UPF1 ATPase, cleaved by the SMG6 endonuclease and degraded by the XRN1 cytoplasmic exonucle-ase. NMD surveillance, however, does not entirely explain the mechanism of decreased STAT3 expres-sion. In addition to exon skipping, ASO treatment causes intron retention and reduction of chromatin-associated STAT3 mRNA. The application of steric blocking ASOs to promote RNA degradation allows one to explore more nucleotide modifications than tolerated by RNase H or RISC-dependent ASOs, with the goal of improving ASO drug properties