Single-molecule visualization of DNA G-quadruplex formation in live cells.


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Authors
Radzevičius, Antanas  ORCID logo  https://orcid.org/0000-0002-4552-2967
Ranasinghe, Rohan T 
Abstract

Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.

Description
Keywords
Cell Line, Tumor, Fluorescent Dyes, G-Quadruplexes, G1 Phase, Humans, Microscopy, Fluorescence, S Phase, Single Molecule Imaging, Time-Lapse Imaging
Journal Title
Nat Chem
Conference Name
Journal ISSN
1755-4330
1755-4349
Volume Title
12
Publisher
Springer Science and Business Media LLC
Rights
All rights reserved
Sponsorship
Cancer Research UK (CB4330)
Cancer Research UK (19836)
Medical Research Council (MR/K015850/1)
Royal Society (RP150066)
Engineering and Physical Sciences Research Council (EP/L027631/1)
Cancer Research UK (18618)
Supported by programme grant funding from Cancer Research UK (C9681/A18618, S.B.) core funding from Cancer Research UK (C14303/A17197, S.B.), a Royal Society University Research Fellowship (UF120277 to S.F.L.), Research Professorship (RP150066 to D.K.), a EPSRC (EP/L027631/1 to D.K.) and a BBSRC David Phillips Fellowship (BB/R011605/1 to M.D.A)