Department of Physics, McGill University, Montreal, QC H3A 2T8, Canada.
Michael Smith Laboratories, University of British Columbia, BC V6T 1Z4, Canada.
Nucleic Acids Res. 2022 Mar 21;50(5):2754-2764. doi: 10.1093/nar/gkac101.
Many cellular processes occur out of equilibrium. This includes site-specific unwinding in supercoiled DNA, which may play an important role in gene regulation. Here, we use the Convex Lens-induced Confinement (CLiC) single-molecule microscopy platform to study these processes with high-throughput and without artificial constraints on molecular structures or interactions. We use two model DNA plasmid systems, pFLIP-FUSE and pUC19, to study the dynamics of supercoiling-induced secondary structural transitions after perturbations away from equilibrium. We find that structural transitions can be slow, leading to long-lived structural states whose kinetics depend on the duration and direction of perturbation. Our findings highlight the importance of out-of-equilibrium studies when characterizing the complex structural dynamics of DNA and understanding the mechanisms of gene regulation.
许多细胞过程都是处于非平衡状态下发生的。这包括在超螺旋 DNA 中的特定部位解旋，这可能在基因调控中发挥重要作用。在这里，我们使用凸面镜头诱导限制（CLiC）单分子显微镜平台，以高通量的方式研究这些过程，而不会对分子结构或相互作用施加人为限制。我们使用两个模型 DNA 质粒系统，pFLIP-FUSE 和 pUC19，来研究在远离平衡态时超螺旋引起的二级结构转变的动力学。我们发现结构转变可能很缓慢，导致存在长寿命的结构状态，其动力学取决于扰动的持续时间和方向。我们的研究结果强调了在描述 DNA 的复杂结构动力学和理解基因调控机制时，进行非平衡态研究的重要性。
