# Dysregulation of the DNA damage response by phosphorothioate antisense oligonucleotides

## TL;DR

This paper shows how certain antisense oligonucleotides disrupt DNA repair processes, leading to toxic DNA damage.

## Contribution

The study reveals that phosphorothioate ASOs trigger DNA repair enzyme activation and condensate formation without DNA damage.

## Key findings

- Phosphorothioate ASOs bind DNA repair enzymes and form nuclear condensates.
- ASO-induced condensates activate DNA damage response and disrupt repair processes.
- This dysregulation leads to toxic DNA lesions and cell cycle checkpoint activation.

## Abstract

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.

Antisense oligonucleotides (ASOs) are used in research and therapy. Here, the authors show that PS-modified ASOs bind key DNA repair enzymes and form nuclear condensates that dysregulate the DNA damage response, disrupt DNA repair, and cause toxic DNA lesions, revealing mechanisms underlying ASO-induced toxicity.