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

Abstract

The G4C2 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 (G2C4)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(G2C4)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.

Figure 1. CD and NMR spectra indicate the presence of protonated structures.

(a) CD spectra of d(G₂C₄) (black), d(G₂C₄)₂ (green), d(G₂C₄)₄ (blue), and d(G₂C₄)₈ (purple) immediately after dilution in H₂O (dashed line) and after incubation (full line). Positive and negative peaks, at 285 and 265 nm, respectively, are associated with the formation of i-motifs. (b) Comparison of CD spectra of d(G₂C₄)₄ in water (dashed lines) and under molecular crowding conditions in 40% w/v PEG8000 (full lines) at pH 6.5 (purple), 7.0 (green), 7.2 (blue). Peaks characteristic for i-motifs persist up to pH 7.0 in 40% w/v PEG8000. All spectra were acquired at 37 °C. (c) Imino region of ¹H NMR spectra of d(G₂C₄), d(G₂C₄)₂, d(G₂C₄)_4, and d(G₂C₄)₈ after incubation in H₂O at pH 6.0 and 5 °C. Signals between δ 12.5 and 13.5 ppm are indicative of Watson-Crick hydrogen bonding, while signals between δ 16.0 and 17.0 ppm correspond to imino protons from hemi-protonated C⁺-C base pairs.

Figure 2. Coexistence of i-motifs and protonated hairpins.

(a) Imino region of ¹H NMR spectra of d(G₂C₄)₄ at 5 °C and pH 4.7, 6.0, and 7.2. (b) Imino region of ¹H NMR spectra of d(G₂C₄)₄ at 5, 25, and 37 °C at pH 4.7. The vertical scale of the spectral region indicated by the dotted box has been enlarged three-fold. (c) Imino region of ¹H NMR spectra of d(G₂C₄)₈ at 5 °C and pH 4.7, 6.0, and 7.2. (d) Imino region of ¹H NMR spectra of d(G₂C₄)₈ at 5, 25, and 37 °C at pH 4.7. The vertical scale of the spectral region indicated by the dotted box has been enlarged three-fold.

Figure 3. Assignment of guanine imino protons and proposed structures.

(a) Imino-imino region of NOESY spectrum of d(G₂C₄)₄ at mixing time of 150 ms. (b) Assignment of imino protons of guanine residues involved in GC base pairs using 1D ¹⁵N-edited HSQC NMR spectra of residue specific (marked on the left side of each spectrum) ¹⁵N, ¹³C-isotopically labelled d(G₂C₄)₄. Top spectrum in B represents imino region of ¹H NMR spectrum. All spectra were acquired at pH 4.7 and 5 °C. (c) Proposed structures of i-motif and (d,e) hairpins adopted by d(G₂C₄)₄.

Figure 4. i-motifs persist under molecular crowding and in the presence of the complementary strand.

(a) Imino region of ¹H NMR spectra of d(G₂C₄)₄ and (b) d(G₂C₄)₈ in water and in 40% w/v PEG8000, at pH 6.0 with 100 mM K⁺ ions. (c) Imino region of ¹H NMR spectra of equimolar mixture of d(G₂C₄)₈ and d(G₄C₂)₈ in water at pH 4.7 and 6.0, and in 40% w/v PEG8000 at pH 6.0 with 100 mM K⁺ ions. The vertical scale of the spectral region indicated by the dotted box has been enlarged five-fold. All spectra were recorded at 37 °C.