Crystal structures of distinct parallel and antiparallel DNA G-quadruplexes reveal structural polymorphism in C9orf72 G4C2 repeats.

The abnormal expansion of GGGGCC (G4C2) repeats in the noncoding region of the C9orf72 gene is a major genetic cause of two devastating neurodegenerative disorders, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These G4C2 repeats are known to form G-quadruplex (G4) structures, which are hypothesized to contribute to disease pathogenesis. Here, we demonstrated that four DNA G4C2 repeats can fold into two structurally distinct G4 conformations: a parallel and an antiparallel topology. The high-resolution crystal structure of the parallel G4 reveals an eight-layered dimeric assembly, formed by two identical monomeric units. Each unit contains four stacked G-tetrads connected by three propeller CC loops and is stabilized through 5'-to-5' π-π interactions and coordination with a central K+ ion. Notably, the 3'-ending cytosines form a C·C+·C·C+ quadruple base pair stacking onto the adjacent G-tetrad layer. In contrast, the antiparallel G4 adopts a four-layered monomeric structure with three edgewise loops, where the C6 and C18 bases engage in stacking interaction with neighboring G-tetrad via a K+ ion. These structurally distinct G-quadruplexes provide mechanistic insights into C9orf72-associated neurodegeneration and offer potential targets for the development of structure-based therapeutic strategies for ALS and FTD.