BioAFM Journal Club

Base-Pair Resolution Analysis of the Effect of Supercoiling on DNA Flexibility and Major Groove Recognition by Triplex-Forming Oligonucleotides

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

A note from one of the authors, Dr. Alice Pyne:

"Our study shows some of the highest resolution images of individual DNA molecules ever taken, and was led by three women in Physics, a rare and fantastic combination."

Nature Communications volume 12, Article number: 1053 (2021)

In this publication, researchers have combined atomic force microscopy (AFM) and computational simulation studies to study DNA supercoiling and the effect it has on the structure and dynamics of DNA. DNA supercoiling is omnipresent in nature and is involved in, e.g., the regulation of transcription, replication, and chromosomal segregation.

In an interdisciplinary collaboration between the groups of Alice Pyne (University of Sheffield), Agnes Noy (University of York), and Sarah Harris (University of Leeds), researchers combined high-resolution AFM and atomistic molecular dynamics (MD) simulations to reveal how negative supercoiling affects global and local DNA conformation, as well as structure and dynamics in canonical B-form DNA minicircles at double-helix resolution. The helical structure, disruptions, defects of supercoiled DNA, and even triplex formation after the addition of triplex-forming oligonucleotides (TFO) were resolved using a MultiMode 8 and Dimension FastScan Bio in PeakForce Tapping mode.

Using high-resolution AFM and in silico data, the direct effect of varying degrees of negative supercoiling (topoisomers) on the minicircle DNA structure could be revealed, e.g., the critical bend angle at which defects, which dominate DNA mechanics, appear. The aspect ratio and radius of gyration were determined, which describe DNA compaction with increasing supercoiling. Finally, the effect of conformational changes on triplex formation with TFO was investigated, revealing the balance between the electrostatic and bonding interactions involved.

The results show how supercoiling determines the conformation, mechanics, and dynamics of DNA, and ultimately, molecular recognition.