Issue 40, 2024

Multiscale topological analysis of kinetoplast DNA via high-resolution AFM

Abstract

Kinetoplast DNA is a complex nanoscale network, naturally assembled from thousands of interconnected DNA circles within the mitochondrion of certain parasites. Despite the relevance of this molecule to parasitology and the recent discovery of tuneable mechanics, its topology remains highly contested. Here we present a multiscale analysis into the structure of kDNA using a combination of high-resolution atomic force microscopy and custom-designed image analysis protocols. By capturing a notably large set of high-resolution images, we are able to look beyond individual kDNA variations and quantify population properties throughout several length scales. Within the sample, geometric fluctuations of area and mean curvature are observed, corresponding with previous in vitro measurements. These translate to localised variations in density, with a sample-wide decrease in DNA density from the outer rim of the molecule to the centre and an increase in pore size. Nodes were investigated in a single molecule study, and their estimated connectivity significantly exceeded mean valence, with a high dependence on their position in the network. While node separation was approximately half the minicircle circumference, it followed a strong bimodal distribution, suggesting more complex underlying behaviour. Finally, upon selective digestion of the network, breakdown of the fibril-cap heterogeneity was observed, with molecules expanding less upon immobilisation on the mica surface. Additionally, preferential digestion was seen in localised areas of the network, increasing pore size disproportionately. Overall, the combination of high-resolution AFM and single molecule image analysis provides a promising method to the continued investigation of complex nanoscale structures. These findings support the ongoing characterisation of kDNA topology to aid understanding of its biological and mechanical phenomena.

Graphical abstract: Multiscale topological analysis of kinetoplast DNA via high-resolution AFM

Supplementary files

Article information

Article type
Paper
Submitted
30 Apr 2024
Accepted
05 Sep 2024
First published
11 Sep 2024
This article is Open Access
Creative Commons BY license

Phys. Chem. Chem. Phys., 2024,26, 25798-25807

Multiscale topological analysis of kinetoplast DNA via high-resolution AFM

B. Diggines, S. Whittle, I. Yadav, E. P. Holmes, D. E. Rollins, T. E. Catley, P. S. Doyle and A. L. B. Pyne, Phys. Chem. Chem. Phys., 2024, 26, 25798 DOI: 10.1039/D4CP01795A

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