Nanopore Studies of DNA Damage and G-quadruplex Folding in the Human Telomere Sequence

The bacterial pore-forming toxin α-hemolysin has dimensions appropriate for capture and translocation of DNA strands in a single-molecule electrophoresis experiment. We used the nanopore's properties to study G-quadruplex unfolding in the human telomere repeat sequence with and without the presence of DNA lesions introduced into either the GGG tracks of a potential G-quadruplex (via oxidation) or the TTA loops (via photodimerization). Different topological folds of the G-quadruplex could be distinguished by either their current-time signatures or by their unfolding rates when a dA25 tail was added. Another more compact four-stranded structure, the i-motif, was also studied and found to be exceptionally well folded and stable inside the nanopore cavity. Comparisons are made between early studies with a home-built nanopore device vs. the currently available instrument from Oxford Nanopore Technologies (ONT), showing that we approach, but do not yet achieve, single-molecule sequencing of DNA damage sites in human telomere repeats. These studies aid in our understanding of the structure and dynamics of non-canonically folded DNA, its behaviour in crowded environments that mimic intracellular conditions, and the ability to use nanopore sequencing to identify DNA damage sites in this oxidation-prone segment of the genome.