Chemically stabilized long antisense oligonucleotides perturb RNA exosome function.

The interaction with intracellular proteins shapes the pharmacology and toxicity of RNA drugs like gapmers and siRNAs. Here, we identify the RNA exosome as a modification-sensitive interaction partner of long, chemically stabilized, single-stranded RNA drugs. Using proximity biotinylation proteomics, we observe preferential binding of the catalytic exosome subunits DIS3 and EXOSC10 to oligonucleotides stabilized by 2'-ribose modifications and phosphorothioate linkages. RNA drug binding correlates with exosome impairment as measured by dose-dependent stabilization of promoter upstream transcripts and accumulation of ribosomal RNA processing intermediates leading to reduced cell viability. Consistent with structural requirements for substrate threading through the exosome channel, oligonucleotides longer than 24 nucleotides can impair exosome function. Systematic variation of ribose and backbone chemistry reveals that exosome inhibition is highly sensitive to the spatial arrangement of chemical modifications and can be mitigated by incorporation of bulky 2'-MOE residues or alternative internucleoside linkages like mesyl phosphoramidate. In contrast to the clinically established RNA drug modalities, class-specific liabilities of emerging ADAR-recruiting oligonucleotides remain poorly understood. Importantly, we demonstrate that ADAR-recruiting oligonucleotides can be designed that preserve editing activity while minimizing exosome interference. These findings illuminate critical variables that must be considered in the safer design of next-generation RNA base editing therapeutics.