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 a very slow setup with pulling velocity and force load accessible to high-speed atomic force microscopy. We identified multiple factors affecting the unfolding mechanism, i.e.,: (i) the more the direction of force was perpendicular to the GQ channel axis (determined by GQ topology), the more the base unzipping mechanism happened, (ii) the more parallel the direction of force was, GQ opening and cross-like GQs were more likely to occur, (iii) strand slippage mechanism was possible for GQs with an all-anti pattern in a strand, and (iv) slower pulling velocity led to richer structural dynamics with sampling of more intermediates and partial refolding events. We also identified that a GQ may eventually unfold after a force drop under forces smaller than those that the GQ withstood before the drop. Finally, we found out that different unfolding intermediates could have very similar chain end-to-end distances, 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

“…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 anisotropic mechanical stability of GQs may be relevant to biological processes, as endogenous forces act on DNA, e.g., by the effect of superhelical coiling or specialized enzymes called helicases. − Therefore, mechanochemical properties of GQs have been studied by single-molecule pulling techniques both experimentally ,,− ,− and computationally. − ,, 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.…”

“…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.…”

“…Magnetic tweezer, optical tweezer, and atomic force microscopy (AFM), which can be further integrated with single-molecule fluorescence resonance energy transfer technique, are the most popular experimental methods that have been used multiple times to investigate folding and unfolding mechanisms of various intramolecular GQs. ,− As a computational analog to experimental pulling methods, steered molecular dynamics (SMD) simulation , drags the system out from an initial configuration by applying the external force. SMD techniques have indeed been applied to investigate the unfolding of the hTel GQs − and the thrombin-binding aptamer …”