%0 Generic
%A Oedegaard Fougner, Oeyvind
%C Heidelberg
%D 2021
%F heidok:30343
%R 10.11588/heidok.00030343
%T A method to image the 3D structure of human genes in single cells with 10 kb resolution
%U https://archiv.ub.uni-heidelberg.de/volltextserver/30343/
%X The spatial organisation of the genome is essential for its functions including gene expression, DNA replication and repair, as well as chromosome compaction and segregation. Below the level of the large linear chromosomal DNA molecules, more compact topologically associating domains (TADs) have been identified as fundamental units of chromosome structure. However, the actual three-dimensional (3D) folding of DNA within TADs still needs to be understood.    Based on theoretical simulations, we predicted that the nanoscale resolving power of super-resolution microscopy can in principle address this key open question. Here, we present the development of an experimental approach that combines super-resolution microscopy with Exchange-PAINT of barcoded in situ hybridisation probes and their computational analysis to extract the 3D path of the linear DNA sequence underlying TADs. We demonstrate that this method can resolve the physical structure of the DNA at a resolution of ~500 bp in vitro and ~10 kb in single human cells. Given the predicted genomic loop sizes and our ability to reconstruct the physical DNA path from the positions of combinatorial in situ hybridisation labels, the experimental and computational pipeline developed in this thesis is ready to be scaled-up to probe the 3D organisation of entire chromosomes at ~10 kb resolution in single human cells.