Abstract
Antisense oligonucleotides (ASOs) are synthetic oligonucleotides that alter expression of disease-associated transcripts via Watson–Crick hybridization. ASOs that function through RNase H or the RNA-induced silencing complex (RISC) result in enzymatic degradation of target RNA. ASOs designed to sterically block access of proteins to the RNA modulate mRNA metabolism but do not typically cause degradation. Here, we rationally design steric blocking ASOs to promote mRNA reduction and characterize 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 skipping 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 exonuclease. NMD surveillance, however, does not entirely explain the mechanism of decreased STAT3 expression. 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.
Highlights
Antisense oligonucleotides (ASOs) are powerful and extremely versatile therapeutic agents utilized in a growing number of applications including RNA reduction, translation arrest, miRNA inhibition, splicing modulation and polyadenylation site selection [1,2]
Modified 2 -MOE ASOs act through steric hindrance and do not support RNase H or RNA-induced silencing complex (RISC) activity
Cells were transfected with 50 nM uniform 2 -MOE ASOs and 24 h posttransfection exon skipping was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR)
Summary
Antisense oligonucleotides (ASOs) are powerful and extremely versatile therapeutic agents utilized in a growing number of applications including RNA reduction, translation arrest, miRNA inhibition, splicing modulation and polyadenylation site selection [1,2]. The most widespread use of ASOs is for decreasing the expression of protein coding RNAs through an RNase H or RNA-induced silencing complex (RISC) mechanism. ASOs containing DNA recruit RNase H to the DNA–RNA heteroduplex, where it cleaves the RNA and promotes subsequent degradation by cellular nucleases [3]. Small interfering RNAs (siRNAs) reduce mRNA expression after assembly of RISC and Argonaute 2 (AGO2) cleavage of the target mRNA [4]. 2 modifications in the central DNA ‘gap’ of an ASO impair RNase H activity and are limited to the 5 and 3 wings of the ASO [6]. Single-stranded and doublestranded siRNAs have unique structural and chemical requirements that limit the repertoire of modifications supportive of AGO2 activity [7,8]
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