Non-equilibrium structural dynamics of supercoiled DNA plasmids exhibits asymmetrical relaxation
Cynthia Shaheen, Cameron Hastie, Kimberly Metera, Shane Scott, Zhi Zhang, Sitong Chen, Gracia Gu, Lisa Weber, Brian Munsky, Fedor Kouzine, David Levens, Craig J. Benham, Sabrina Leslie
Abstract
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.