BPS2026 – Elucidating the folding and unfolding dynamics of ALS and FTD-associated G4C2 microsatellite repeat RNA using molecular simulations

An intronic G4C2 hexanucleotide repeat in the C9orf72 gene is the major genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Expanded G4C2 RNA directly contributes to disease pathology and has emerged as a potential target for small molecule and anti-sense oligonucleotide (ASO) therapeutics. Hence, understanding the structural dynamics of G4C2 RNA is a crucial step toward the development of RNA-targeting therapies. Here, using equilibrium and non-equilibrium all-atom molecular dynamics (MD) simulations, we explored potential intermediates in the de novo folding path of the G4C2 repeat sequence of different sizes. Starting with an unfolded A-form single-stranded RNA structure, G4C2 undergoes an ensemble of conformations, illustrating various higher order topologies, it can adapt, resemble hairpin, knot, and higher order structures like G-quadruplex (GQ). Next, using a crystallized G4C2 repeat RNA structure in GQ conformation, we performed equilibrium MD simulation and steered molecular dynamics (SMD) simulation to characterize its stability, structural fluctuations, and the unfolding pathways. Our results suggest two distinct mechanisms of G4C2 RNA GQ unfolding: unzipping and strand slippage. Taken together, our findings reveal a molecular level understanding of G4C2 RNA structure, dynamics, limitations of the current state of MD simulation studies for GQ studies, and provide a platform for further small-molecule RNA interaction studies.