F Nuclear Magnetic Resonance Fingerprinting Technique for Identifying and Quantifying G-Quadruplex Topology in Human Telomeric Overhangs.

G-quadruplexes (G4s) are noncanonical nucleic acid secondary structures with diverse topological features and biological roles. Human telomeric (Htelo) overhangs consisting of TTAGGG repeats can fold into G4s that adopt different topologies under physiological conditions. These G4s are potential targets for anticancer drugs. Despite intensive research, the existence and topology of G4s at Htelo overhangs in vivo are still unclear because there is no method to distinguish and quantify the topology of Htelo overhangs with native lengths that can form more than three tandem G4s in living cells. Herein, we present a novel F chemical shift fingerprinting technique to identify and quantify the topology of the Htelo overhangs up to five G-quadruplexes (G4s) and 120 nucleotides long both in vitro and in living cells. Our results show that longer overhang sequences tend to form stable G4s at the 5'- and 3'-ends, while the interior G4s are dynamic and "sliding" along the sequence, with TTA or 1-3 TTAGGG repeats as a linker. Each G4 in the longer overhang is conformationally heterogeneous, but the predominant ones are hybrid-2, two- or three-tetrad antiparallel, and hybrid-1 at the 5'-terminal, interior, and 3'-terminal, respectively. Additionally, we observed a distinct behavior of different lengths of telomeric sequences in living cells, suggesting that the overhang length and protein accessibility are related to its function. This technique provides a powerful tool for quickly identifying the folding topology and relative population of long Htelo overhangs, which may provide valuable insights into telomere functionality and be beneficial for structure-based anticancer drug development targeting G4s.