An antiparallel G-quadruplex structure with a 5′-end overhang forms predominantly at the human telomeric ds–ss DNA junction

G-quadruplex structures formed by human telomeric sequences are highly sensitive to subtle sequence variations and local environment. While most structural studies have focused on sequences containing four uninterrupted G-tracts, the structural consequences of splitting a G-tract remain unexplored. Here, we present the NMR solution structure of a human telomeric sequence with the first G-tract split. The structure adopts an antiparallel topology with a disordered GTT segment at the 5′-end. This conformation is preferentially formed at a biologically relevant double-strand/single-strand DNA junction. These findings broaden our understanding of the structural diversity of human telomeric G-quadruplexes.