Project description
Real-time imaging of DNA G-quadruplexes
DNA sequences rich in guanine nucleotides form tetrameric structures known as G-quadruplexes with potential biological roles in telomere function and maintenance, replication and transcription. However, we lack the tools to visualise G-quadruplexes directly in live cells. To address this issue, the EU-funded PLIM-G4 project proposes to develop optical probes that interact with different DNA topologies and upon doing so, their emission lifetime changes in a topology-dependent manner. Optimisation of the properties of these probes will enable the real-time imaging of G-quadruplexes in live cells, allowing to unveil novel information on the role and the dynamics of G-quadruplexes in biological processes.
Objective
There has been increasing experimental evidence suggesting that tetra-stranded DNA structures (G-quadruplexes) play important biological roles in telomere function and maintenance, replication and transcription. The most direct evidence for their existence has come from immuno-staining in fixed cells as well as from recent deep sequencing studies. However, to date, we are still lacking tools that allows us to visualizing G-quadruplexes directly in live cells. While several small-molecule probes that fluoresce upon interaction with DNA have been reported, none of them have been successful at imaging G-quadruplexes in a cellular environment. This is mainly due to the fact that they rely on changes in intensity which are not possible to track properly in a cellular environment. Recently, the host lab reported a novel strategy to image G-quadruplexes in live cells. This approach makes use of the changes in emission lifetime (rather than intensity) of optical probes upon their interaction with different topologies of DNA. Since life-time is concentration independent, this approach can be successfully used to image G-quadruplexes in live cells. While this has proven to be a highly successful approach, it is still in its infancy since the probe developed so far has a number of limitations such as low brightness, relatively small lifetime range and low selectivity. Thus, this project aims to develop a new set of probes that address all these issues and use them to image the dynamics of G-quadruplexes in live cells in real time. I propose to develop platinum complexes (which 'switch-on' their phosphorescent upon interactions with DNA) with high affinity and selectivity for G-quadruplexes. To achieve this, novel approaches for automated synthesis and high-throughput analysis will be developed. The new probes will be used to carry out Phosphorescence Lifetime Imaging Microscopy (PLIM) studies to give evidence for the first time of the dynamics of G-quadruplexes in live cells.
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Programme(s)
Funding Scheme
MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Coordinator
SW7 2AZ LONDON
United Kingdom