Abstract
Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). 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 G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s 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 G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.
Highlights
Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s)
We previously reported that DAOTA-M2 [Fig. 1a] has a very different fluorescence lifetime when bound to G4 structures as compared to duplex or single-stranded DNA, with good live cell permeability and low cytotoxicity[32]
We demonstrate that DAOTA-M2 responds to the expression of DNA helicases FancJ and RTEL1 which are involved in genome stability and the distribution of G4 in live cells
Summary
Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). The prevalence of G4s in human chromatin has been investigated using an immunoprecipitation technique[9,10] These studies showed that there are over 10,000 sequences in the human genome that can form G4 DNA structures under cellular conditions[9]. Subsequent studies have reported high-affinity antibodies able to visualise G4 DNA and G4 RNA in mammalian cells by immunofluorescent staining[16,17,18]. While these elegant studies are the most direct evidence of the presence of G4s in cells, they have some potential drawbacks. The fixation process can denature the cellular DNA and induce DNA degradation[20,21], and the high affinity of the antibodies for G4 could artificially increase the presence of
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
