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Single-molecule G-quadruplex visualisation in live cells and 3D chromatin


Type

Thesis

Change log

Authors

Radzevicius, Antanas  ORCID logo  https://orcid.org/0000-0002-4552-2967

Abstract

The G-quadruplex (G4) is a secondary DNA structure formed by the self-assembly of guanine nucleotides. Mapping G4 formation in the human genome shows that they are enriched in functionally important regions such as promoters, enhancers, transcription factor and architectural protein binding sites. However, the roles of G4s in biology remains elusive. Whilst G4s can exist in a folded or unfolded state, methods are required to ensure that endogenous G4 detection does not perturb G4 folding dynamics. In this work, G4-specific fluorescent probes are reported which enable single-molecule real-time visualisation of G4 structures in living cells. Single-molecule imaging was performed at nanomolar probe concentrations providing measurements of relative populations of G4s in living cells without global perturbation of G4 dynamics. Chemical trapping of the unfolded G4 state reveals that G4s alternate between their folded and unfolded state over a time course of 20 minutes. G4 formation in live cells was also shown to be cell-cycle dependent and disrupted by inhibition of transcription and replication. The observations presented here provide evidence to support dynamic formation of G4s in living cells.

G4 visualisation methods were then deployed with the aim of investigating the potential involvement of G4s in 3D chromatin organisation. For this, G4 imaging was extended into 3D by super-resolution microscopy in fixed cells, and combined with single-cell Hi-C method. Hi-C is a fixed nucleus chromatin conformation capture technique that can simulate 3D chromatin structure via high-throughput sequencing of DNA strands in close proximity. 3D imaging of G4s has revealed their localisation throughout the nucleus volume with several regions showing clusters. When G4 mapping data were overlapped with single-cell Hi-C structures and G4s were visualised in 3D chromatin, this showed that G4s co-localised with the A compartment of the nucleus. By overlapping Hi-C structures with 3D super-resolved G4 images of the same single cells and population derived G4 sequencing data, the aim of this work is to establish a foundation to understand G4 relationships with respect to higher order 3D chromatin structure.

Part of this work has been published: M. Di Antonio, A. Ponjavic, A. Radzevicius, R. T. Ranasinghe, M. Catalano, X. Zhang, J. Shen, L.-M. Needham, S. F. Lee, D. Klenerman, and S. Balasubramanian, ”Single- molecule visualization of DNA G-quadruplex formation in live cells,” Nat. Chem., vol. 12, pp. 832-837, 2020.

Description

Date

2022-03-30

Advisors

Balasubramanian, Shankar

Keywords

G-quadruplex, Single-molecule fluorescence imaging, 3D STORM, Super-resolution imaging, Hi-C, Genome architecture, Nucleic acids, Chemistry, Biophysics, Antibody conjugation, Organic synthesis

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
Cambridge Trust, Herchel Smith fund