Abstract
<jats:p>The single-stranded G-overhangs of vertebrate telomeres, composed of guanine (G)-rich hexanucleotide repeats, can adopt six register-dependent permutations: GGTTAG, GTTAGG, TTAGGG, TAGGGT, AGGGTA, and GGGTTA. Telomeric G-quadruplexes (G4s) formed by the first two repeat permutations, which are the most prevalent at human chromosome ends, have been largely overlooked. Here, G4 formation across all six permutations, each containing 4 to 9 repeats, are systematically investigated under varying cation conditions and molecular crowding environments. Our findings reveal that distinct repeat permutations yield markedly different G4 stabilities and folding topologies, which in turn modulate DNA polymerase activity. This expanded perspective on telomeric structural diversity provides new insights into telomere biology and suggests that multiple repeat patterns may cooperatively influence chromosome end protection and targeting.</jats:p>