Stable Off-Path Structures In The Folding Dynamics Of Two Consecutive Telomeric Dna G-Quadruplexes

G-quadruplexes are non-canonical DNA structures able to form in guanine-rich DNA sequences. Hundred thousands of potential and confirmed G-quadruplex forming regions have been identified throughout the human genomic DNA (Huppert and Balasubramanian NAR 2005; Chambers et al. Nat. Biotechnol. 2015; Bedrat, Lacroix, and Mergny NAR 2016). Amongst those sequences, many are able to support formation of multiple consecutive G-quadruplexes. One such region is the ∼300 nucleotide long repeating (TTAGGG) overhang of the mammalian telomere. Formation of G-quadruplexes has been shown to block important DNA modifying enzymes and the G-quadruplex interphase is a potential drug target. Using single molecule FRET TIRF microscopy with alternating laser excitation, we investigated folding of multiple G-quadruplex structures and identified distinct conformational stages taken up by the constructs in the presence of various monovalent cations. Our results show a large conformation diversity in the presence of Na+ and K+. In experiments with Na+, where antiparallel G-quadruplex formation was favored, we found that time-traces could be sorted into two distinct non-interconverting population stages. Only <3.5% time-traces was observed in which both population stages were visited indicative of a high interconversion energy barrier. The observed populations included several conformations of the fully folded structures and structures with only one antiparallel G-quadruplex. Some of these very stable conformations block full folding and thus are off the folding pathway. Taken together, our observations suggest a model where G-quadruplexes form randomly along the telomeric overhang in a way that sometimes block full condensation.