Non-equilibrium structural dynamics of supercoiled DNA plasmids exhibits asymmetrical relaxation

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.

Figure 1.

Oligo-binding assay for plasmid state determination. (A) Schematic of Convex Lens-induced Confinement (CLiC) microscopy. A flow cell is created by adhering two coverslips with double-sided tape. Micrometer-sized pits were etched into the bottom coverslip using lithographic techniques. The top coverslip is depressed with a convex lens to trap molecules in the pits for prolonged observation. For illustrative purposes, a mean value of one plasmid per pit is shown, though in experiments on average there are 23 plasmids and 0.82 oligos per pit. (B) The supercoiled plasmid is modeled as transitioning between an open O and closed state C. This is a simplification which assumes that the unwinding site is the only structure present in the plasmid. Estimates of the opening and closing rates are implicitly affected by the presence of other structures, indicated here by a question mark. (C) An oligonucleotide can bind to an open site to form a bound oligo-plasmid complex. Oligo unbinding was so slow that for the purposes of our experiments, this reaction was assumed to be irreversible. (D) Approximately 400 pits can be observed in a single field of view. Bound oligos are characterized by their slower diffusion relative to free oligos. Circles indicate pits containing bound oligos. The total number of bound oligos is counted in each video. Inset shows pits containing 0, 1, 2 or 3 bound oligos. (E) The number of bound oligo-plasmid complexes averaged over 10 videos as a function of time of the plasmid pFLIP-FUSE (σ = −0.10 ± 0.01) interacting with a 20 bp long oligonucleotide at a series of temperatures. Error bars represent the standard error of the mean. Plasmids were kept at 4°C prior to the start of the experiment, when they were mixed with oligo and brought to the experimental temperature.

Figure 2.

The number of fluorescent oligos bound to pFLIP-FUSE (σ = −0.062 ± 0.006) molecules per 400 pits, averaged over 10 videos, as a function of pre-experiment temperature treatment. (A) pFLIP-FUSE plasmids were heated from 4 to 37°C and held at this temperature for either 0, 3 or 144 h prior to the addition of the oligo and oxygen scavengers at t = 0 s. (B) Plasmids were heated to 95°C for 1 min and cooled to 37°C at a rate of 5°C/min to prevent mismatching. Plasmids were then held at 37°C for either 0, 3, 23 or 144 h, prior to the addition of the oligo and oxygen scavengers at t = 0 min. The ‘no heating’ curve is the 0 h curve from the graph in (A) and is included for scale. Brackets indicate areas of the same scale between (A) and (B). Fits are generated using the parameters in Table 1. All error bars represent the standard error of the mean. Top traces indicate the temperature of the plasmids. The decrease to room temperature just before time 0 indicates when the sample was transferred from the thermal cycler to the microscope.

Figure 3.

The number of fluorescent oligonucleotides bound to pUC19 (σ = −0.054 ± 0.006) molecules per 400 pits, averaged over 10 videos, with respect to different pre-experiment temperature treatments. (A) pUC19 plasmids were held at 95°C for either 1, 30, 60 or 120 min before being cooled to 37°C at a rate of 5°C/min prior to the addition of the oligo and oxygen scavengers at t = 0 min. (B) Plasmids were heated to 95°C for 60 min and cooled to 37°C at a rate of 5°C/min. Plasmids were then held at 37°C for either 0, 3 or 20 h, prior to the addition of the oligo and oxygen scavengers at t = 0 min. The 'no heating' curve on both graphs is pUC19 that had been heated directly to 37°C prior to the addition of the oligo. Curves are fitted using the parameters in Table 1. All error bars represent the standard error of the mean. Top traces indicate the temperature of the plasmids. The decrease to room temperature just before time 0 indicates when the sample was transferred from the thermal cycler to the microscope.

Figure 4.

Secondary structures present in the plasmids pFLIP-FUSE (σ = −0.10 ± 0.01, (A and B)) and pUC19 (σ = −0.097 ± 0.009, (C and D)). (A) Schematic illustrating the location of the FUSE region and a cruciform region in pFLIP-FUSE with respect to the NcoI-HF cut site and origin of replication (Ori). (C) Schematic illustrating the location of the two unwinding sites in pUC19 with respect to the HindIII cut site and origin of replication (Ori). (B and D) Potassium permanganate footprinting analysis illustrating secondary structures present in pFLIP-FUSE (B) or pUC19 (D) when directly heated to 37°C (left lane, -) or heated to 95°C and cooled to 37°C (right lane, +). After heating, plasmids were treated with potassium permanganate, then cut with either NcoI-HF (pFLIP-FUSE) or HindIII (pUC19) followd by an S1 nuclease treatment. The S1 nuclease cut the plasmids at the structures indicated in (A) and (C), if they were present.