With new probes, scientists can see how four-stranded DNA interacts with molecules in living human cells and clarifies their role in cellular processes. DNA usually forms the classic double helix shape of two strands wrapped around each other. While DNA can form more exotic shapes in test tubes, few are seen in real living cells.
However, four-stranded DNA known as G-quadruplex has recently been observed to form naturally in human cells. Well, in new research published in today Nature communicationA team led by scientists from Imperial College London has developed new probes that can be used to see how G-quadruplexes interact with other molecules in living cells.
G-quadruplexes are found in higher concentrations in cancer cells, so it is believed that they play a role in the disease. The probes show how G-quadruplexes are "unwound" by certain proteins and can also help identify molecules that bind to G-quadruplexes, leading to potential new drug targets that can perturb their activity.
One of the lead authors, Ben Lewis of the Department of Chemistry at Imperial, said, "Another form of DNA will have a huge impact on all processes involved – like reading, copying, or expressing genetic information. The fact that G-quadruplexes play an important role play an important role in the mapping of this structure directly into living cells. "
G-quadruplexes are rare in cells, which means that standard techniques for detecting such molecules have difficulty detecting them specifically. Ben Lewis describes the problem as "like finding a needle in a haystack, but the needle is also made of hay".
To solve the problem, researchers from the Vilar and Kuimova groups in Imperial's Department of Chemistry teamed up with the Vannier group of the London Institute of Medical Sciences of the Medical Research Council.
They used a chemical probe called DAOTA-M2 that fluoresces (lights up) in the presence of G-quadruplexes, but instead of monitoring the brightness of the fluorescence, they monitored how long that fluorescence lasted. This signal is not dependent on the concentration of the probe or the G-quadruplexes, which means it can be used to clearly visualize these rare molecules.
Dr. Marina Kuimova of Imperial's Department of Chemistry said, "By using this more nuanced approach, we can remove the difficulties that have prevented the development of reliable probes for this DNA structure."
The team used their probes to study the interaction of G-quadruplexes with two helicase proteins – molecules that "unwind" DNA structures. They showed that when these helicase proteins were removed, more G-quadruplexes were present, which shows that the helicases play a role in the unwinding and thus in the breakdown of G-quadruplexes.
Dr. Jean-Baptiste Vannier of the MRC London Institute of Medical Sciences and the Institute of Clinical Sciences at Imperial said: "In the past we had to rely on studying indirect evidence of what these helicases were doing, but now let's look at them directly in living cells. "
They also studied the ability of other molecules to interact with G-quadruplexes in living cells. When a molecule introduced into a cell binds to this DNA structure, it displaces the DAOTA-M2 probe and shortens its lifespan. how long the fluorescence lasts.
In this way, interactions in the nucleus of living cells can be examined and more molecules, for example those that do not fluoresce and are not visible under the microscope, can be better understood. Professor Ramon Vilar of the Department of Chemistry at Imperial stated, "Many researchers have been interested in the potential of G-quadruplex binding molecules as potential drugs for diseases such as cancer. Our method will help improve our understanding of these potential new drugs."
Peter Summers, another lead author for the Department of Chemistry at Imperial, said, "This project has been a fantastic opportunity to work at the intersection of chemistry, biology and physics. Without the expertise and close collaboration of all three, this would not have been possible Research groups. "
The three groups intend to continue working together to improve the properties of their probe, investigate new biological problems, and further shed light on the role of G-quadruplexes in our living cells. The research was funded by the Imperial Excellence Fund for Frontier Research.