Involvement of Long-Lived Intermediate States in the Complex Folding Pathway of the Human Telomeric G-Quadruplex

The energy landscapes of human telomeric G-quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real-time NMR spectroscopy, we investigated the K+-induced folding of the human telomeric DNA sequence 5'-TTGGG(TTAGGG)(3)A-3'. Three long-lived states were detected during folding: a major conformation (hybrid-1), a previously structurally uncharacterized minor conformation (hybrid-2), and a partially unfolded state. The minor hybrid-2 conformation is formed faster than the more stable hybrid-1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin-like structures.