Abstract
Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.
Highlights
Friedreich’s ataxia (FRDA) is the most common inherited autosomal recessive ataxia, and in >96% of cases the disease is correlated by expansion of (GAA)n repeats in the first intron of the Frataxin gene (FXN) [1,2]
In a previous study on the formation of triplex structures at FRDA (GAA)n repeats, we showed that a low-molecular weight benzoquinoquinoxaline compound (BQQ), recognizes triplex structures formed at (GAA)n repeats in plasmids
We previously demonstrated that homopyrimidine natural DNA triplex forming oligonucleotides (TFOs) bind dsDNA at global structural changes in plasmids containing (GAA) repeat sequences forming a pyrimidine motif triplex
Summary
Friedreich’s ataxia (FRDA) is the most common inherited autosomal recessive ataxia, and in >96% of cases the disease is correlated by expansion of (GAA)n repeats in the first intron of the Frataxin gene (FXN) [1,2]. Disease-associated expanded alleles consist of approximately 70 to more than 1000 repeats [1,2,3]. The (GAA)n expansions result in a substantial reduction in Frataxin mRNA and protein levels [4], and non-symptomatic carriers (heterozygous for the expanded allele) show ~50% reduction [1,3,4,5]. Several studies have focused on increasing the level of Frataxin through targeting the epigenetic regulation of gene expression. There are no FRDA therapeutics available, only symptomatic management strategies
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