Thermally activated irreversible homogenization of G-quadruplexes in an ALS/FTD-associated nucleotide expansion

A significant proportion of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases exhibit a substantial copy number expansion of the hexanucleotide GGGGCC/GGCCCC sequence in the C9ORF72 gene. The GGGGCC sequence forms a non-canonical 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. We further show that this reaction is irreversible, since subsequent temperature sweeps do not show CD shifts from non-parallel to parallel G4 topologies. 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 non-canonical DNA structures may play a role in cellular homeostasis or disease pathogenesis. SIGNIFICANCEThe GGGGCC repeats in the C9ORF72 gene expand in copy number in certain neurodegenerative diseases, forming non-canonical DNA structures called G-quadruplexes (G4) which are associated with the pathological state. However, why the repeat expansion occurs is not known, and a key may lie in the thermodynamic stability of certain G4 conformations. Here, we use CD spectroscopy to experimentally report thermally activated heat-induced G4 conformation homogenization, from a heterogeneous population to the parallel configuration. We derive an analytical biophysical theory which is compatible with this experimental observation, which is shown to be irreversible. Our in vitro tuning of the free energy landscape that modulates G4 conformational fidelity motivates a search for possible in vivo enzymatic regulators.