The structure of DNA is called a double helix, which looks like a twisted staircase. While DNA can form some more exotic shapes in test tubes, few are seen in real living cells.
Recently, scientists at the Imperial College London have discovered the formation of four-stranded DNA in human cells. Scientists have created new probes that can see how G-quadruplexes are interacting with other molecules inside living cells.
Four-stranded DNA, known as G-quadruplex, is usually found in higher concentrations in cancer cells; hence scientists think that G-quadruplexes play a role in the disease. Further examination reveals that G-quadruplexes are ‘unwound’ by specific proteins and help identify molecules that bind to G-quadruplexes, leading to potential new drug targets that can disrupt their activity.
Lead authors Ben Lewis, from the Department of Chemistry at Imperial, said: “A different DNA shape will have an enormous impact on all processes involving it – such as reading, copying, or expressing genetic information.
“Evidence has been mounting that G-quadruplexes play an important role in a wide variety of processes vital for life, and a range of diseases, but the missing link has been imaging this structure directly in living cells.”
G-quadruplexes are uncommon inside cells, which means standard methods for recognizing such molecules experience issues identifying them explicitly. Ben Lewis describes the problem as “like finding a needle in a haystack, yet the needle is additionally made of hay.
To solve the problem, analysts from the Vilar and Kuimova groups in the Department of Chemistry at Imperial collaborated with the Vannier group from the Medical Research Council’s London Institute of Medical Sciences.
Using a chemical probe called DAOTA-M2, which fluoresces (lights up) in the presence of G-quadruplexes, but instead of monitoring the brightness of fluorescence, they observed how long this fluorescence lasts. This signal does not depend on the probe’s concentration or G-quadruplexes, meaning it can be used to visualize these rare molecules unequivocally.
Dr. Marina Kuimova, from the Department of Chemistry at Imperial, said: “By applying this more sophisticated approach, we can remove the difficulties which have prevented the development of reliable probes for this DNA structure.”
Using the probes, scientists later studied G-quadruplexes’ interaction with two helicase proteins – molecules that ‘unwind’ DNA structures. They showed that if these helicase proteins were removed, more G-quadruplexes were present, indicating that the helicases play a role in unwinding and breaking down G-quadruplexes.
Scientists also studied the ability of other molecules to interact with G-quadruplexes in living cells. If a molecule introduced to a cell binds to this DNA structure, it will displace the DAOTA-M2 probe and reduce its lifetime, i.e., how long the fluorescence lasts.
This allows interactions to be studied inside the nucleus of living cells and for more molecules, such as those which are not fluorescent and can’t be seen under the microscope, to be better understood.
From the Department of Chemistry at Imperial, Professor Ramon Vilar explained: “Many researchers have been interested in the potential of G-quadruplex binding molecules as potential drugs for diseases such as cancers. Our method will help to progress our understanding of these potential new drugs.”
Peter Summers, another lead author from the Department of Chemistry at Imperial, said: “This project has been a fantastic opportunity to work at the intersection of chemistry, biology, and physics. It would not have been possible without the expertise and close working relationship of all three research groups.”
- Summers, P.A., Lewis, B.W., Gonzalez-Garcia, J. et al. Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy. Nat Commun 12, 162 (2021). DOI: 10.1038/s41467-020-20414-7