Folding pathways and force-induced unfolding of neurodegeneration associated GGGGCC microsatellite repeat RNA...

An intronic G4C2 hexanucleotide repeat expansion in the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). G4C2 RNA itself directly contributes to disease mechanisms and has emerged as a potential target for small molecules, anti-sense oligonucleotides (ASOs), and CRISPR-based therapeutics. Hence, understanding the folding/unfolding and structural polymorphism is essential for G4C2 RNA-targeting therapies. Here, using equilibrium all-atom molecular dynamics (MD) simulations, we explored potential intermediate metastable conformations of the G4C2 RNA repeats and investigated the effect of repeat length on folding. G4C2 RNA undergoes an ensemble of intermediate metastable states resembling hairpin, knot, and a G-quadruplex (GQ) like structures. Enhanced torsional flexibility and conformational heterogeneity were observed with increasing repeat length. Next, using a crystallized G4C2 RNA structure in GQ conformation, we performed equilibrium MD simulations to reveal its thermodynamic stability. Steered molecular dynamics (SMD) simulations with a reduced model of G4C2 GQ uncover two distinct unfolding mechanisms along the chosen reaction coordinates: strand slippage and unzipping. Overall, our findings provide molecular-level insights into the folding and force-induced unfolding dynamics of G4C2 repeat RNA GQ and set a platform for future studies on small-molecule targeting of ALS/FTD-associated G4C2 RNA.