G-quadruplex formation of oligonucleotides containing ALS and FTD related GGGGCC repeat

Abstract

A largely increased number of GGGGCC repeats located in the non-coding region of C9orf72 gene have been identified as the leading cause of two related neurological disorders, familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We examined G-quadruplex forming ability of GGGGCCrepeat containing oligonucleotides with four guanine tracts chosen as the smallest possible model to form a unimolecular G-quadruplex. These oligonucleotides are readily to folded into G-quadruplexes in the presence of K+ ions. However, the formation of multiple structures makes structural analysis challenging and time consuming. We observed that flanking sequences on 5'- and 3'-ends as well as mutations of loop residues have a profound effect on folding. Sequence d[(G4C2)3G4] was chosen for further scrutiny and optimization of nuclear magnetic resonance (NMR) spectroscopic properties with dG to 8Br-dG substitutions at specific positions in the sequence under different folding conditions. Expectedly, folding into desired predominant topology is facilitated when substituted residue adopted a syn conformation in the naturally-occurring structure. Single dG to 8Br-dG substitution at position 21 and fine tuning of folding conditions facilitate folding of d[(G4C2)3GGBrGG] into (mostly) a single G-quadruplex, and thus enable determination of its high-resolution structure by high-field NMR.