Dysregulation of the DNA damage response by phosphorothioate antisense oligonucleotides

Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) are widely used to modulate gene expression in basic research and therapy. Within cells, these ASOs seed nuclear structures with unclear functions and consequences. At DNA breaks, endogenous nucleotide polymers drive the assembly of biomolecular condensates that recruit repair proteins, but the underlying mechanism(s) and effects on repair enzyme activation are poorly understood. Here, we show that ASOs bind to DNA-PKcs, ATM, and PARP1, triggering phase separation and formation of nuclear condensates containing ASOs and these essential repair enzymes. Condensates assembly is stimulated by ASO concentration and ATM activity, while limited by DNA-PKcs activity. Notably, these condensates become enzymatically active and erroneously elicit the DNA damage response in the absence of DNA damage, activating cell cycle checkpoints, disturbing endogenous repair and causing accumulation of toxic DNA lesions. These findings uncover mechanisms for ASO toxicity and the activation of DNA repair enzymes by nucleotide polymers.