Fast MAS NMR Spectroscopy Can Identify G-Quartets and Double-Stranded Structures in Aggregates Formed by GGGGCC RNA Repeats

The expansion of GGGGCC repeats within the C9orf72 gene has been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In neurons from patients with expanded repeats, C9orf72 GGGGCC repeat RNA predominantly forms nuclear foci, and in vitro, repeat-containing RNA can self-aggregate. Two structural motifs have been proposed to provide the interstrand interactions that drive aggregation: G-quartet (G4) structures and double-strand interactions with GG mismatches. Using in vitro transcribed RNA with physiologically relevant number of repeats, we were able to form gel-like aggregates suitable for investingation using fast MAS NMR spectroscopy. This approach enabled us to characterize the dominant interstrand interactions within the RNA gels. Both Watson–Crick and Hoogsteen base pairs were identified in RNA gels formed by RNA with 48 GGGGCC repeats. Their relative abundance shifted upon reconstitution in the presence of different divalent cations or nuclear extracts, underscoring the dynamic equilibrium between G-quadruplex and duplex interactions in GGGGCC RNA aggregation.