Anja Kovanda (1), Matja Zalar (2), Primož Šket (2,3), Janez Plavec (2,3,4), Boris Rogelj (1)
(1) Department of Biotechnology, Institute Jozef Stefan, Ljubljana, Slovenia
(2) Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia
(3) EN-FIST Center of Excellence, Ljubljana, Slovenia
(4) Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
The intronic G4C2 hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most common mutation associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The pure GC content of this mutation enables the formation of special non-B DNA structures such as G-quadruplexes and i-motifs that can act as promoters and regulatory elements affecting replication, transcription and translation of the surrounding region. C-rich sequences, similar to the anti-sense (G2C4)n strand may form i-motifs consisting of two parallel duplexes in a head to tail orientation held together by hemi-protonated C+ -C pairs under acidic conditions, however, whether such structures can form from the antisense strand under more physiological conditions remains unknown. Using CD and NMR we show for the first time that d(G2C4)n repeats do form i-motif and protonated hairpins even under near-physiological conditions. Furthermore, when both strands are present, rather than forming a DNA duplex, G-quadruplex and i-motif/hairpin structures preferentially form. This phenomenon could explain the replicational and transcriptional instability of this mutation and the i-motifs/hairpin structures could represent a novel pharmacological target for C9ORF72 associated ALS and FTLD.