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Luminescent probe finds rare four-helix DNA in living human cells



Luminescent probe finds rare four-helix DNA in living human cells

Four-helix DNA.Image source: Imperial College London

The new probe enables scientists to see the interaction of four-stranded DNA with molecules in living human cells, thereby revealing its role in cellular processes.

DNA usually forms the classic double helix shape of two strands entangled together. Although DNA can form more peculiar shapes in test tubes, it is rarely seen in actual living cells.

However, it has recently been discovered that four-stranded DNA (called G-quadruplex) is naturally formed in human cells.Now, in the new research published today Nature CommunicationsA research team led by scientists at Imperial College London has created a new probe that can observe how the G-quadruplex interacts with other molecules in living cells.

The concentration of G-quadruplexes in cancer cells is high, so it is thought to play a role in this disease. These probes reveal how certain proteins unravel the G-quadruplex and can also help identify molecules that bind to the G-quadruplex, leading to new drug targets that may destroy their activity.

Needle in a Haystack

One of the lead authors of the Department of Chemistry at Imperial College, Ben Lewis, said: “Different DNA shapes will have a huge impact on all processes involved, such as reading, copying or expressing genetic information.

“More and more evidences show that G-quadruplexes play an important role in various processes that are vital to life and many diseases, but the missing link has always been directly related to this structure in living cells. For imaging.”

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G-quadruplexes are rare in cells, which means that standard techniques for detecting such molecules are difficult to detect specifically. Ben Lewis described the problem as “it’s like finding a needle in a haystack, but the needle is also made of hay.”

To solve this problem, researchers from the Vilar and Kuimova groups of the Department of Chemistry at Imperial College collaborated with the Vannier group of the London Institute of Medical Sciences of the Medical Research Council.

Luminescent probe finds rare four-helix DNA in living human cells

Fluorescence lifetime imaging micrograph of nuclear DNA in living cells stained with the new probe. The color indicates that the fluorescence lifetime is between 9 (red) and 13 (blue) nanoseconds.Image source: Imperial College London

They used a chemical probe called DAOTA-M2, which fluoresces (lights up) in the presence of the G quadruplex, but they do not monitor the brightness of the fluorescence, but monitor the fluorescence duration. This signal does not depend on the concentration of the probe or G-quadruplex, which means it can be used to clearly observe these rare molecules.

Dr. Marina Kuimova of the Department of Chemistry at Imperial College London said: “By adopting this more complex method, we can eliminate the difficulties that hinder the development of reliable probes for the DNA structure.”

See directly in living cells

The team used their probes to study the interaction between the G-quadruplex and two helicase proteins (molecules that “unwind” the DNA structure). They showed that if these helicase proteins were removed, more G-quadruplexes would be present, indicating that helicases play a role in melting and thus destroying G-quadruplexes.

Dr. Jean-Baptiste Vannier from MRC’s London Institute of Medical Sciences and Imperial College’s Institute of Clinical Sciences said: “In the past, we had to rely on observing the indirect signs of the action of these helicases, but now we look directly in living cells they.”

They also studied the ability of other molecules to interact with G-quadruplexes in living cells. If the molecule introduced into the cell binds to the DNA structure, it will replace the DAOTA-M2 probe and shorten its lifetime, that is, how long the fluorescence lasts.

This allows for a better understanding of the interactions inside the nucleus of living cells, as well as more molecules, such as molecules that do not fluoresce and are invisible under the microscope.

Professor Ramon Vilar from the Department of Chemistry at Imperial College London explained: “Many researchers are interested in the potential of G-quadruplex binding molecules as potential drugs to treat diseases such as cancer. Our method will help. To increase our understanding of these potential new diseases. Drugs.”

Peter Summers, another lead author of the Department of Chemistry at Imperial College, said: “This project is an excellent opportunity to work at the intersection of chemistry, biology and physics. Without the expertise and expertise of experts Close working relationship, this is impossible. Three research groups.”

The three groups intend to continue to collaborate to improve the performance of their probes, explore new biological problems, and further clarify the role of G-quadruplexes in our living cells. The research was funded by the Imperial Research Excellence Fund.


Designer molecules focus on the mysterious four-stranded DNA


More information:
Peter Summers (Peter A. Visualize G-quadruplex DNA dynamics in living cells through a fluorescence lifetime imaging microscope, Nature Communications (2021). DOI: 10.1038 / s41467-020-20414-7

Provided by Imperial College London



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