Complexity of Guanine Quadruplex Unfolding Pathways Revealed by Atomistic Pulling Simulations

Guanine quadruplexes (GQs) are non-canonical nucleic acid structures involved in many biological processes. GQs formed in single-stranded regions often need to be unwound by cellular machinery, so their mechanochemical properties are important. Here, we performed steered molecular dynamics simulations of human telomeric GQs to study their unfolding. We examined four pulling regimes, including very slow setup with pulling velocity and force load accessible to high-speed atomic force microscopy. We identified multiple factors affecting the unfolding mechanism. The more the direction of force was perpendicular to the GQ channel axis (determined by GQ topology), the more the base unzipping mechanism happened. If the GQ had either all-anti or all-syn pattern in a strand, strand slippage mechanism was more likely to occur. Importantly, slower pulling velocity led to richer unfolding pathways including partial refolding attempts. We show that GQ may eventually unfold after force drop under forces smaller than those the GQ withstood before the drop. This suggests that proteins in vivo might resolve GQs even if their stall forces are smaller than GQ rupture force. Finally, we found out that different unfolding intermediates may have very similar chain end-to-end distance, which reveals some limitations of structural interpretations of single-molecule spectroscopic data.