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

Alice L B Pyne,Andrew D Bates,Clare E M Stevenson,Fiorella M Cugliandolo,Anthony Maxwell,Bart W Hoogenboom,Agnes Noy,Lesley A Mitchenall,Joseph G Beton,Sarah A Harris,Michael M Piperakis,Kavit H S Main,Victor Velasco-Berrelleza

doi:10.1038/s41467-021-21243-y

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.

Highlights

In the cell, DNA is arranged into highly-organised and topologically-constrained structures
High-resolution atomic force microscopy (AFM) images recorded in aqueous solution show DNA minicircles, isolated with native levels of supercoiling, in a range of conformations with sufficient resolution to resolve the two oligonucleotide strands of the double helix
The deviation from planarity of the minicircles was calculated to be

Summary

Introduction

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 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 combine high-resolution atomic force microscopy (AFM) with molecular dynamics (MD) simulations to reveal how supercoiling affects global and local DNA conformation, structure and dynamics in DNA minicircles of length 250–340 bp These minicircles are small enough to be simulated at the atomistic level by MD13,19 and to be visualised at high (double-helix) resolution by AFM experiments in solution[20,21,22]. The DNA minicircles in this study incorporate a TFO-binding sequence, to assess how the interplay of electrostatic and base-stacking energies determines the formation of triplex structures in supercoiled DNA

Methods

Results

Conclusion