Organisation and dynamics of individual DNA segments in topologically complex genomes

Capturing the physical organisation and dynamics of genomic regions is one of the major open challenges in biology. The kinetoplast DNA (kDNA) is a topologically complex genome, made by thousands of DNA (mini and maxi) circles interlinked into a two-dimensional Olympic network. The organisation and dynamics of these DNA circles are poorly understood. In this paper, we show that dCas9 linked to quantum dots (QD) can efficiently label DNA maxicircles and different classes of DNA minicircles in kDNA. We use this method to study the distribution and dynamics of different classes of DNA minicircles within the network. We discover that maxicircles display a preference to localise at the periphery of the network and that they undergo subdiffusive dynamics. By using simulations, we discover that this peripheral localisation of maxicircles may contribute to the bucking of the structure in solution. Finally, by tracking the dynamics of the QDs we can also quantify the effective network stiffness, confirming previous indirect estimations via AFM. Our method could be used more generally to quantify the location, dynamics, and material properties of genomic regions in other complex genomes, such as that of bacteria, and to study their behaviour in the presence of DNA-binding proteins.