Long-lived optical probes to image G-quadruplex DNA in live cells

DNA is a versatile biomolecule which can assemble into a wide range of different non-canonical structures beyond the well-known double helix. The recent advances in genomics have revealed that DNA sequences with the potential to form such non-canonical structures (e.g. hairpins, triplexes, quadruplexes, junctions) are widespread in genomes. Interestingly, their distribution is not random: they are selectively enriched in specific genes and particularly in transcriptional regions, hinting at a role for non-canonical DNA structures in essential biological functions. It is now recognised that the link between 'primary structure' (i.e. DNA sequence) and the observed phenotypes needs to be complemented by an understanding of the roles that 'secondary' and 'tertiary' structures of DNA play in vivo.
Amongst these non-canonical DNA structures, G-quadruplexes - which form when guanine-rich DNA sequences fold into quadruply-stranded helices - have received increasing attention over the past 10 years. This is mainly due to the increasing experimental evidence suggesting that G-quadruplex DNA structures play essential biological roles such in telomere function and maintenance, replication and the regulation of gene expression. However, to date there is still little direct evidence that G-quadruplexes form in live cells and are functional.
Based on our preliminary studies in this project we propose to develop new small-molecule optical probes to visualize G-quadruplexes in live cells by Phosphorescence Lifetime Imaging Microscopy (PLIM). In particular, we aim to study the formation and dissolution of G-quadruplexes in the telomeres. Such probes will provide invaluable information about the biological function of these non-canonical DNA structures as well as, in the long term, be useful molecular tools for cellular screening of potential drugs that target quadruplexes.

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 recet 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, two of the applicants 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 use the proof-of-concept studies described above 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. We propose to develop platinum complexes (which 'switch-on' their phosphorescent upon interactions with DNA) with high affinity and selectivity for G-quadruplexes. The new probes will allow us to carry out detailed Phosphorescence Lifetime Imaging Microscopy (PLIM) studies to give evidence for the first time of the dynamics of G-quadruplex formation/dissolution at telomeres in live cells.

There is increasing recognition that non-canonical DNA structures play very important roles in essential biological processes. On the basis of this supposition, many labs, funding bodies and pharmaceutical companies are pursuing and funding research to identify modulators of these unusual topologies. One such non-canonical DNA form is the G-quadruplex, whose presence is proposed to be essential in regulating gene expression, in controlling replication initiation and telomere maintenance. However, the visualisation and imaging of such DNA structures in live cells is still to be achieved. Our project aims to develop new molecular tools to image G-quadruplex DNA in live cells. This will provide for the first time the possibility of studying the formation/resolution of these non-canonical DNA structures in real time and hence understand their dynamics at different stages of the cell cycle as well as their interplay with proteins (in particular helicases). Furthermore, this information could also provide important clues as to whether G-quadruplexes can be targeted by small molecule and hence are 'drugable' targets. Therefore, the proposed research will have an impact at different levels. In the short term, it will provide important fundamental understanding of quadruplexes in live cells (particularly their dynamics in relation to DNA-molecule and DNA-protein interactions). We envisage that by the end of our project, the new probes will be available (e.g. via a chemical company such as Invitrogen) to the wider academic community so that the involvement of G-quadruplexes in a wide range of biological processes can be ascertained.
This in turn will have an important long-term impact in the search for drugs that target this type of non-canonical DNA structure since our probes could provide the basis for a cellular assay to screen small molecules against quadruplexes.
In addition to their use as DNA probes, the new molecules prepared in this project will have photophysical properties which could be potentially used for other applications such as sensing and optoelectronic materials. Indeed, platinum complexes have been previously self-assembled into functional materials with optoelectronic properties.
Two other important impacts of this research are: a) Training of a young researcher in a highly multidisciplinary environment including synthetic chemistry/chemical biology and cell biology; b) Outreach to broader scientific community and non scientists - by using optical probes (visually very appealing and easy to relate to) we will probe DNA structures beyond the well-known canonical DNA double helix. We believe that this can easily capture the public's imagination and therefore the project is well suited for outreach activities.