Visualizing structure-mediated interactions in supercoiled DNA molecules

Abstract

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid's structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

Figure 1.

DNA supercoiling promotes unwinding. (A) Supercoiled plasmid DNA experiences stress due to either over- or under-twisting of the DNA double helix. The torsional stress caused by this can force the DNA double helix to cross over itself, or affect the average number of base pairs (bp) per complete turn of the double helix, A = 10.4 bp/turn. (B) Sufficiently high stress allows for the structural transitions along the molecule, such as DNA unwinding. Here, n bases are single-stranded, or unwound, and wrap about each other by a certain number of radians per base, τ. (C) DNA probe molecules bind to target sites only after they become unwound, allowing for visualization of unwinding.

Figure 2.

CLiC nanopit microscopy and binding events. (A) Schematic of the CLiC method confining plasmids and labeled oligo-probes to pits made using micro/nano lithography techniques (28). The circle in the front-center pit demonstrates a pit containing a bound probe–plasmid complex. (B) Schematic of plasmids and a freely diffusing probe confined to a circular pit of 3-μm diameter and 0.5-μm depth. Each pit contains ∼24 plasmids and a single labeled probe. (C) Fluorescence image of mixture of probes and pUC19 plasmids with mean superhelical density <σ> = 0 and standard deviation s_σ = 0.03 confined to pits. ( D) Fluorescence image of mixture of probes and pUC19 plasmids with superhelical density <σ> = − 0.050, s_σ = 0.07 confined to pits. The gray square denotes the two pits containing single probes in F and G. (E) Fluorescence image of mixture of probes and pUC19 plasmids with <σ> = − 0.127, s_σ = 0.06 confined to pits. White circles show binding events. (F) Pit containing a bound probe–plasmid complex from the data set shown in D. (G) Pit containing a freely diffusing probe from the data set shown in D. Scale bars in C–E denote 10 μm, while those in F and G are 3 μm. Images in C–G had their background subtracted using a rolling ball algorithm with a radius of 50 pixels, and a Gaussian-blur filter with radius 0.5 pixels was applied to each.

Figure 3.

Partitioning of supercoiling. (A) A completely relaxed plasmid molecule. There are 12 turns of the double helix, and, as the helix does not cross over itself, the twist (Tw) is equal to 12 and writhe (Wr) is equal to 0. (B) The same plasmid molecule as in a), but supercoiled. The double helix has been untwisted by 2 turns but does not cross over itself, leading to Tw = 10 and Wr = 0. ( C) The supercoiled molecule in b can release some of the undertwisting stress energy by crossing the double helix over itself. Here, Tw = 12 while Wr = −2. (D) Another means of relieving stress energy is through site unwinding, depicted here for the same plasmid as in B and C. Whether a plasmid partitions its energy into Tw, Wr, or other secondary structures, such as unwinding, is probabilistic for each base pair, and depends on its local energy landscape.

Figure 4.

Statistical mechanics predictions of unwinding. DZCBtrans and SIDD profiles for mean superhelical density <σ> = −0.120 with standard deviation s_σ = 0.007 as a function of temperature for the Site 1 region of pUC19. Inset: schematic of Site 1 unwinding.

Figure 5.

probe–plasmid binding and temperature. (A) The number of binding events per 100 probes averaged over 10 different videos vs. time, for three temperatures studied. Curves represent fits to the data. The plasmids have a superhelical density of <σ> = −0.108, s_σ = 0.07. Inset: Reaction rates vs. temperature obtained from the data fits. (B) The number of unwound plasmids obtained from the fits in a) per 100 probes vs. temperature. Inset: the short-time reaction rate, k_s, versus temperature for the data in part A. The reaction rates are fit to an exponential demonstrating Arrhenius behavior. Each data point was averaged over ∼3300 probes, indicating high-throughput. Error bars in (A) and the inset of (B) denote the standard error of the mean of the binding for the 10 averaged videos on the y-axis, while those on the x-axis represent the standard error of the mean of the time. Error bars on the fit parameters represent the 95% confidence interval of the fit.

Figure 6.

Oligo-plasmid binding and superhelical density. (A) Number of binding events per 100 probes averaged over 10 different videos versus time, for a series of <σ>. Inset: Reaction rates versus <σ> obtained from the data fits. (B) The number of unwound plasmids obtained from the fits in (A) per 100 probes versus <σ>. Inset: the ‘short-time reaction rate,’ k_s, versus <σ> for the data in part A. All samples presented here have s_σ = 0.007. Error bars in (A) and the inset of (B) denote the standard error of the mean of the binding for the 10 averaged videos on the y-axis, while those on the x-axis represent the standard error of the mean of the time. Error bars on the fit parameters represent the 95% confidence interval of the fit.

Figure 7.

Oligo-plasmid binding and oligo sequence. (A) Number of binding events per 100 probes averaged over 10 different videos vs. time for 3 different probe sizes: 20 bases (black), 30 bases (dark gray), and 75 bases (light gray). Inset: schematic of the area on the unwinding region where each probe binds. (B) Number of binding events per 100 probes averaged over 10 different videos versus time for two different probe sequences: probe targeting the center (black), and probe targeting the side (dark gray). Inset: schematic of the area on the unwinding region where each probe binds. (C) Probe nucleotide sequences for the ( i) 20-base probe, (ii) 30-base edge probe, (iii) 30-base center probe and (iv) 75-base probe. (v) Part of the pUC19 unwinding sequence (underlined), and the wound bases adjacent to this region (not underlined). Error bars on the y-axis denote the standard error of the mean of the binding for the 10 averaged videos, while those on the x-axis represent the standard error of the mean of the time.

Figure 8.

Oligo-plasmid binding capture event. (A) The spatial variance in intensity vs. time for a probe/plasmid binding event. This event was captured for a plasmid with <σ> = −0.151 and s_σ = 0.007 at T = 31°C. A median filter with a 10-frame window was applied to suppress noise. (B) Images selected at a series of observation times. Background noise was subtracted using a rolling ball algorithm with a 50 pixel radius. Scale bars denote 2.5 μm.

Figure 9.

Plasmid-probe unbinding initiated by topoisomerase IA. Percentage of pits containing bound complexes over time for a solution containing 752 pM of the 30-base pair probe and 21.1 nM of pUC19 in 20 mM tris, 50 mM potassium acetate, and 100 μg/mL BSA after the addition of 28.48 μg/mL of E. coli topoisomerase IA. Topoisomerase IA was added at t = 0.