Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides

Abstract

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

Fig. 1. Structural and dynamic diversity in supercoiled DNA minicircles.

a–d High-resolution AFM images of natively supercoiled (σ = 0.03–0.06) DNA minicircles of 251 bp (a) and 339 bp (b–d) showing their helical structure and disruptions of canonical B-form DNA (marked by red arrowheads), where the angle of the helix changes rapidly, or where the DNA appears thinner or disrupted. Aspect ratios for each molecule: 048 (a), 0.44 (b bottom), 0.87 (b top), 0.78 (c) and 0.65 (d). e MD snapshots of minicircle conformations for 251 (first image) and 339 bp corresponding to the minicircles in the AFM images selected by visual inspection from explicitly solvated simulations (first, second and third images at ΔLk −1, 0 and −2, respectively) and from implicitly solvated simulations (fourth and fifth image) at ΔLk = 0. Top and side views (top and bottom row, respectively) show the degree of planarity of the depicted structures, where top refers to the top view of adsorbed DNA minicircles, and side the perpendicular plane. White and red lines indicate plectonemic loops of 9 and 6.5 nm width, respectively (see ‘Methods’). Aspect ratios are 0.45 ± 0.04, 0.30 ± 0.03, 0.86 ± 0.01, 0.81 ± 0.01 and 0.69 ± 0.01. f Time-lapse AFM measurements of a natively supercoiled 339 bp DNA minicircle, recorded at 3 min/frame. Fast scan direction is shown by white arrows. g Chronological snapshots from simulations of 500 ps duration for a 339 bp minicircle with ΔLk = −1 (see Supplementary Videos 6 and 7). Scale bars (inset): 10 nm and height scale (inset, d): 2.5 nm for all AFM images.

Fig. 2. Supercoiling induces defect formation in 339 bp DNA minicircles, while increasing writhe and compaction.

a MD average structures showing increased defect formation at higher supercoiling, the numbers at the top of each figure are ΔLk for each structure. b Bending calculation obtained by the SerraLINE program using the WrLINE profile from the −3 topoisomer trajectory, where bend angles are calculated as a directional change in tangent vectors separated by 16 bp (additional bending profiles in Supplementary Fig. 3). All peaks >35° are classified as B-DNA bends (black cross) or defects (red triangles) depending on whether canonical non-bonded interactions were broken. c Determination of bending angles in natively supercoiled DNA by high-resolution AFM (white lines), scale bar: 10 nm and height scale 2.5 nm. d Bent-DNA analysis of DNA minicircles by high-resolution AFM (natively supercoiled, first column), and MD simulations (topoisomers 0 to −6, a) shows a  ≈ 75° cut-off between B-DNA (black crosses) and defects (red triangles), with an increase of the latter with supercoiling. e Radius of gyration (Rg) and writhe for the different topoisomers extracted from MD simulations. Grey shading (b) corresponds to standard deviations.

Fig. 3. Negative supercoiling induces global compaction of DNA minicircles, with a conformational change observed at physiological levels of supercoiling.

a AFM images of DNA minicircle populations show increased writhe and compaction at increased negative superhelical density. Images are processed to obtain individual minicircles (red) for analysis. Height scale (inset): 4 nm and scale bar: 50 nm. b Five percent TAC acrylamide gel of negatively supercoiled topoisomers of 339 bp (ΔLk from −1 to −4.9) generated by the addition of increasing amounts of ethidium bromide during the re-ligation reaction. ΔLk = −4.9 is taken from a separate gel image. N = nicked minicircle; R = relaxed minicircle; markers (left-hand lane) are low molecular weight DNA ladder from NEB (sizes from bottom are: 25, 50, 75, 100, 150, 200, 250, 300, 350 and 500 bp). c Representative images of 339 bp minicircles for a range of superhelical densities showing increased levels of compaction and defects (observed as regions of high bending angle, or discontinuities in DNA structure, marked by red arrowheads) for highly supercoiled minicircles. Height scale (inset, a): 4 nm and all images are 80 nm wide. d The relationship between minicircle aspect ratio and supercoiling as a Kernel Density Estimate (KDE) plot of the probability distribution for each topoisomer (N = 1375). e The relationship between minicircle aspect ratio and supercoiling shown as a violin plot for each minicircle topoisomer.

Fig. 4. Conformational diversity in supercoiled DNA minicircles contributes to the triplex formation.

a AFM images showing triplex formation across a range of DNA minicircle conformations. Triplex regions are visible as small, sub-nanometre protrusions from the DNA marked by green arrowheads. Height scales (scale bar inset): 3 nm and scale bars: (single minicircles) 10 nm and (population): 50 nm. b Representative structures of DNA triplex from −6 and +1 topoisomer simulations compared to linear DNA. Arrows indicate less favourable Hoogsteen hydrogen bonds in positively supercoiled DNA. The WC-pyrimidine strand is erased from ΔLk = +1 image for visualisation purposes. c Violin plot of non-bonded interactions for the triplex-binding site (ΔE_bind;L), showing the relative contributions from in-plane base interactions (e.g. WC and Hoogsteen hydrogen bonds) (green), compared to interactions between adjacent bases (e.g. bifurcated and backbone hydrogen bonds and stacking energies) (blue). d Violin plot for electrostatics of the whole minicircle (ΔE_elec;0) with (orange) and without (purple) TFO bound. e Minicircle writhe for modelled topoisomers with (orange) and without (purple) TFO bound. Inset shows a half helical turn reduction in writhe on triplex binding for the ΔLk = −6 topoisomer.