The discovery of the DNA double helix was revolutionary because it demonstrated the structure and function of DNA are intrinsically linked. The double helix is not the only structure DNA can form at physiological conditions, however. The G-quadruplex (G4) is able to form in G-rich sequences of DNA, which evidence suggests plays a crucial role in a range of biological processes. The direct imaging of this structure inside live cells has, to-date, proved difficult.
In 2015, DAOTA-M2 was first reported – a triangulenium dye which discriminated between G4 and non-G4 DNA topologies using fluorescence lifetime and could be used for live cell imaging. Here, we take this probe and use complementary methods to verify that it is, in fact, able to specifically image G4 DNA in cellulo without interference from other biomolecules. We then apply DAOTA-M2 to explore questions about the biological handling of G4 DNA by helicase proteins and directly observe how the presence of two G4-associated helicases is linked to the prevalence of G4 DNA in live cells.
Whilst DAOTA-M2 has many qualities, it is an imperfect probe – suffering especially from low quantum yield. We explore how alterations to the DAOTA-M2 structure can change its properties. First, we examine divergent alterations to the fluorescent core of the molecule with BDATA-M2 and CDATA-M2 and develop new insight into the subtle thermodynamics which allow this class of G4 probes to function. Second, we explore how altering the substituents of the molecule impact its performance with (C3M)2-DAOTA and (C2M)(C3M)-DAOTA, which prove themselves as improved second generation triangulenium probes for G4 DNA. These are used to explore further biological phenomena.
Finally, we observed an unexpected phenomenon in our studies – a sensitivity to viscosity through the fluorescence lifetimes of these dyes. We characterise that response and perform preliminary studies using trianguleniums to sense viscosity.