Complexity of Guanine Quadruplex Unfolding Pathways Revealed by Atomistic Pulling Simulations

Abstract

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.

Mechanical Stability and Unfolding Pathways of Parallel Tetrameric G-Quadruplexes Probed by Pulling Simulations

“…Unzipping is the main (and initial) unfolding transition under all the setups, but the whole spectrum of observed structural transitions is comparable to the pulling of intramolecular GQs reported by us earlier. 107 We observed substantial quantitative differences in rupture forces among the GQ systems and tested force directions, which means that the response of the GQ stem to the pulling force is anisotropic. Overall, the simulations confirm that at the atomic resolution, the unfolding of GQ under external force consists of a series of structural transitions rather than being a sudden one-step rupture of the structure.…”

“…This was somewhat surprising, since the importance of strand slippage in (un)folding of parallel stranded GQs was suggested in several preceding studies. 77,92,106,107 In this work, most of the strand slippage events occurred when the spring-anchor strand had only two Gs attached to the GQ body, requiring less rebuilding of the H-bonds network. This result does not mean that strand slippage is unimportant for GQ (un)folding in the absence of external force, e.g., in thermal unfolding.…”

“…139−145 Therefore, mechanochemical properties of GQs have been studied by single-molecule pulling techniques both experimentally 95,97,100−102,146−154 and computationally. [105][106][107]155,156 The tetrameric GQs studied here likely do not have any direct biological relevance; nevertheless, a link between the models and endogenous GQs can be drawn. The 3Q tetrameric GQ model is a basic scaffold of many biochemically relevant intramolecular parallel-stranded GQs, which differ from the 3Q model only in the presence of propeller loops.…”

“…The OPC favors more unzipping, whereas the SPC/ E (and TIP3P 133 ) water model makes the strand slippage somewhat more likely, though G-unzipping is still the first conformational transition (except for one SPC/E simulation in which the unfolding started directly by strand slippage). This effect of the water model would explain why in previous standard MD, 77,92,134 enhanced-sampling MD, 135 as well as SMD simulations, 106,107 strand slippage was quite frequent for parallel-stranded GQs (some with loops) in SPC/E and TIP3P water; on the other hand, in OPC spontaneous base unzipping (unbinding) occurred even under no force. 78 The differences in the transition preferences between the water models may originate from the different water-base H-bonding and solvation interactions (indirectly affecting the stability of base−base H-bonding and stacking) in the OPC and SPC/E water models.…”

“…Overall, despite a few differences, the unfolding of the tetrameric GQs by external force shows structural richness comparable to the intramolecular telomeric GQ. 107 Role of Spiral-Like Structures. In standard MD runs starting from partially unfolded GQs, unzipping, incorporation of unzipped Gs, and strand slippage were also frequently observed.…”