Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Author:

Summers Peter A.ORCID,Lewis Benjamin W.,Gonzalez-Garcia Jorge,Porreca Rosa M.,Lim Aaron H.M.,Cadinu Paolo,Martin-Pintado Nerea,Mann David,Edel Joshua B.,Vannier Jean BaptisteORCID,Kuimova Marina K.,Vilar Ramon

Abstract

ABSTRACTGuanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) can identify G4 within nuclei of live and fixed cells. We present a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4 and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4 in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

Publisher

Cold Spring Harbor Laboratory

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