Keynote Talk  - Friday, 17 September I 14:00 PM (CEST)

Understanding how DNA behaves in its cellular environment is a challenge of complexity, which can be enhanced by a better understanding of the fundamental properties of DNA. In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. We overcome these limitations by combining high-resolution AFM¹ and atomistic MD simulations to resolve the structure, conformation and dynamics of supercoiled DNA to the base-pair level (Figure 1)².

We use DNA minicircles, only twice the persistence length of DNA, to probe the structure and function of negatively-supercoiled DNA. These minicircles are small enough to be  simulated at the atomistic level by MD and to be visualized at high (double-helix) resolution by AFM experiments in solution 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 flexibility³. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. 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.

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

Figure 1.  Structural diversity in supercoiled DNA minicircles. Synergistic high-resolution AFM images (a-d) and MD snapshots (e) of natively supercoiled DNA minicircles show striking structural diversity in natively supercoiled DNA minicircles. Scale bars: 10 nm. Height scale (inset): 2.5 nm for all images

 

References:

1.           Pyne, A., Thompson, R., Leung, C., Roy, D. & Hoogenboom, B. W. Single-Molecule Reconstruction of Oligonucleotide Secondary Structure by Atomic Force Microscopy. Small 10, 3257–3261 (2014).

2.           Pyne, A. L. B. et al. Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides. Nature Communications 12, 1053 (2021).

3.           Beton, J. G. et al. TopoStats – A program for automated tracing of biomolecules from AFM images. Methods (2021) doi:10.1016/j.ymeth.2021.01.008.