Anti-sense DNA d(GGCCCC)n expansions in C9ORF72 form i-motifs and protonated hairpins

The G(4)C(2) hexanucleotide repeat expansion mutation (HREM) in C9ORF72, represents the most common mutation associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Three main disease mechanisms have been proposed to date: C9ORF72 haploinsufficiency, RNA toxicity, and accumulation of dipeptide repeat proteins. Pure GC content of the HREM potentially enables the formation of various non-B DNA structures such as G-quadruplexes and i-motifs. These structures are proposed to act as promoters and regulatory elements affecting replication, transcription and translation of the surrounding region. G-quadruplexes have already been shown on the G-rich sense DNA and RNA strands (G4C2)(n), the structure of the anti-sense (G(2)C(4)) n strand remains unresolved. Similar C-rich sequences may, under acidic conditions, form i-motifs consisting of two parallel duplexes in a head to tail orientation held together by hemi-protonated C+-C pairs. We show that d(G(2)C(4)) n repeats do form i-motif and protonated hairpins even under near-physiological conditions. Rather than forming a DNA duplex, i-motifs persist even in the presence of the sense strand. This preferential formation of G-quadruplex and i-motif/hairpin structures over duplex DNA, may explain HREM replicational and transcriptional instability. Furthermore, i-motifs/hairpins can represent a novel pharmacological target for C9ORF72 associated ALS and FTLD.