Conformational dynamics of RNA G4C2 and G2C4 repeat expansions causing ALS/FTD using NMR and molecular dynamics studies

G(4)C(2) and G(2)C(4) repeat expansions in chromosome 9 open reading frame 72 (C9orf72) are the most common cause of genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), or c9ALS/FTD. The gene is bidirectionally transcribed, producing G(4)C(2) repeats [r(G(4)C(2))(exp)] and G(2)C(4) repeats [r(G(2)C(4))(exp)]. The c9ALS/FTD repeat expansions are highly structured, and structural studies showed that r(G(4)C(2))(exp) predominantly folds into a hairpin with a periodic array of 1 x 1 G/G internal loops and a G-quadruplex. A small molecule probe revealed that r(G(4)C(2))(exp) also adopts a hairpin structure with 2 x 2 GG/GG internal loops. We studied the conformational dynamics adopted by 2 x 2 GG/GG loops using temperature replica exchange molecular dynamics (T-REMD) and further characterized the structure and underlying dynamics using traditional 2D NMR techniques. These studies showed that the loop's closing base pairs influence both structure and dynamics, particularly the configuration adopted around the glycosidic bond. Interestingly, r(G(2)C(4)) repeats, which fold into an array of 2 x 2 CC/CC internal loops, are not as dynamic. Collectively, these studies emphasize the unique sensitivity of r(G(4)C(2))(exp) to small changes in stacking interactions, which is not observed in r(G(2)C(4))(exp), providing important considerations for further principles in structure-based drug design.