Large-scale expansions of Friedreich′s ataxia GAA·TTC repeats in human cells are prevented by LNA-DNA oligonucleotides and PNA oligomers

Anastasia Rastokina, Negin Mozafari, Jorge Cebrian, Edvard Smith, Sergei M. Mirkin, Rula Zain; bioRxiv 2022.07.04.498742; doi:10.1101/2022.07.04.498742 

The human disease Friedreich′s ataxia (FRDA) is caused by expansions of GAA·TTC repeats in the first intron intron of the frataxin (FXN) gene, and both intergenerational and somatic expansions are crucial for disease development. We and others have shown earlier that expanded GAA·TTC repeats can form an intramolecular triplex structure (H-DNA). Here we studied the effects of locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers on the expansion of GAA·TTC repeats in cultured human cells. Our experimental system employes a mammalian/yeast shuttle plasmid containing a selectable cassette to detect repeat expansions. Using our in-house in vitro triplex-specific DNA cleavage assay, we first confirmed H-DNA formation by the (GAA)100·(TTC)100 repeat in the selectable cassette and demonstrated that the designed LNA-DNA oligonucleotides as well as PNA oligomers are able to disrupt this structure. We then found that both LNA-DNA mixmers and PNA oligomers prevent repeat expansions in human cells. In the accompanying paper, we show that expansions of GAA·TTC repeats in this experimental system occur during replication fork stalling, regression and restart at the repetitive run. We hypothesize, therefore, that triplex DNA formation by the GAA·TTC repeats is a key to their instability, while LNA-DNA oligonucleotides and PNA oligomers counteract repeat expansions by disrupting the triplex at the fork or preventing triplex formation upon fork reversal.