Thermally activated history-dependent homogenization of G-quadruplexes in an ALS/FTD-associated gene

A significant proportion of familial amyotrophic lateral sclerosis and frontotemporal dementia cases exhibit a substantial copy number expansion of the hexanucleotide GGGGCC/GGCCCC sequence in the C9ORF72 gene. The GGGGCC sequence forms a noncanonical DNA structure called a G-quadruplex (G4), which has been associated with the disease states and with nucleic acid condensate formation. G4s can fold into various topologies, which can differentially impact fidelity of DNA synthesis. However, how G4 conformational heterogeneity and its regulation impact hexanucleotide repeat expansion is unclear, and important clues may lie in the thermodynamic properties of different G4 topologies. Here, we use temperature-swept CD spectroscopy to observe configurational homogenization of an initially heterogeneous population of G4s over a small range of temperatures, demonstrating thermally activated behavior. The G4s adopt the parallel configuration after the temperature sweep, and subsequent temperature sweeps show little to no reversal back to nonparallel topologies, suggesting the homogenization is history-dependent. Finally, we provide an analytical theory based on a two-state thermodynamic model which is compatible with experimental evidence, and we discuss alternate mechanisms for the homogenization transition. These findings suggest that kinetic regulation of noncanonical DNA structures may play a role in cellular homeostasis or disease pathogenesis.