Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

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.

Cis- and Trans-Modifiers of Repeat Expansions: Blending Model Systems with Human Genetics

“…However, the lasting effectiveness of this therapy may still be limited by ongoing somatic expansions. Perhaps this therapy will prove effective in combination with treatments designed to disrupt secondary structures[129], slow somatic expansions [81] or promote somatic contractions [130,131].…”

Targeted Oligonucleotides for Treating Neurodegenerative Tandem Repeat Diseases

“…Furthermore, we found that sequence-specific binding using repeat-specific PNA oligomers resulted in complete disruption of the triplex formed and LNA ONs showed similar results (Fig. 5 ) [ 42 ]. These findings are currently employed in FRDA patient cell lines to upregulate FXN transcription and increase the levels of mRNA and protein (manuscript in preparation).…”

“…Furthermore, PNA binds to dsDNA and forms double- and triple-strand structures (Fig. 3 ) through Watson-Crick, Hoogsteen, or reverse-Hoogsteen base pairing [ 42 ]. PNA [ 43 ] and LNA [ 44 , 45 ] can invade dsDNA and form different PNA:DNA, and LNA:DNA, complexes, respectively (Fig.…”

Oligonucleotide Binding to Non-B-DNA in MYC

“…Reports have shown that PNA can both prevent and induce the formation of G-quadruplexes [ 47 ]. Bergquist et al, have demonstrated that PNA and LNA ONs can, by strand-invasion, disrupt H-DNA structures within expanded triplet-repeats, present in various diseases, such as in Friedreich’s ataxia [ 48 ]. To increase the stability of PNA- or LNA-based DNA complexes, clamp type strand-invading ONs have been developed, so called bisPNA and bisLNA, respectively.…”