Imaging G‑Quadruplex\nNucleic Acids in Live\nCells Using Thioflavin T and Fluorescence Lifetime Imaging Microscopy

Visualization of guanine-rich oligonucleotides that fold\ninto G-quadruplex\n(G4) helical structures is of great interest in cell biology. There\nis a large body of evidence that suggests that these noncanonical\nstructures form <i>in vivo</i> and play important biological\nroles. A promising recent development highlighted fluorescence lifetime\nimaging microscopy (FLIM) as a robust technique for the direct and\nquantitative imaging of G4s in live cells. However, this method requires\nspecialized, bespoke synthetic dyes that are not widely available.\nHerein, we demonstrate that the fluorescence lifetime of commercially\navailable environmentally sensitive dyes Thioflavin T (<b>ThT</b>) and Thiazole Orange (<b>TO</b>) is strongly dependent on\nthe type of DNA topology they bind to, with G4s showing long and distinctive\ndecay times that should allow G4 detection in the biological environment.\nWe applied this observation to visualize G4s in live U2OS cells using\nFLIM of <b>ThT</b>, upon alteration in G4 levels due to competitive\nbinding or nuclease treatment of cells.