Confocal microscopy helps reveal presence of four-stranded DNA in living cells
A new fluorescent molecule, when examined using confocal microscopy, can reveal the presence of four-stranded DNA in living cells.
Researchers at Imperial College London (England) are working to understand the four-stranded DNA structures thought to play a role in regulating how genes are switched on and off. To accomplish this, they have created a fluorescent molecule that, when examined using confocal microscopy, can reveal the presence of these structures in living cells.
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DNA is typically arranged in a double helix, where two strands are intertwined like a coiled ladder, but previous research has shown the existence of unusual DNA structures called quadruplexes, where four strands are arranged in the form of little knots.
The team, led by Dr. Marina Kuimova and Prof. Ramon Vilar, used the glowing molecule to target quadruplex DNA inside human bone cancer cells grown in the laboratory. Together with colleagues from Kings College London, they studied the interactions between the two in real time using confocal microscopy.
Quadruplexes can form when a strand of DNA rich in guanines—one of the four building blocks in DNA—folds over onto itself. Several distinct quadruplex structures have been found in the human genome, but their exact role remains unclear. Recent studies have shown they are particularly prevalent in regions nearby oncogenes—genes that have the potential to cause cancer.
|Structure of a G-quadruplex DNA highlighting one of the guanine tetrads. (Credit: Imperial College London)|
"If this can be proved, it would make quadruplexes an extremely important target for treating diseases such as cancer," Vilar says. "But to understand what role they play, we need to be able to study them in living cells. Our new fluorescent molecule allows us to do this by directly monitoring the behavior of quadruplexes inside living cells in real time."
The team designed the fluorescent molecule to glow more intensely when attached to DNA. Using confocal microscopy, they discovered that they could distinguish between the molecules binding to the more common double helical DNA and quadruplex DNA because it glowed for much longer when bound to quadruplexes.
|Confocal microscopy image of the new probe inside human bone cancer cells. The image illustrates the distribution of the probe within the cell and in particular highlights nuclear localization (red and green regions). (Credit: Imperial College London)|
The researchers were also able to visualize the fluorescent molecule being displaced from quadruplex DNA by another molecule known to be a very good quadruplex binder. This suggests that the newly developed molecule could be used to hunt for new compounds that can bind to quadruplexes.
"Until now, to image quadruplexes in cells, researchers have had to hold the cells in place using chemical fixation," says study co-author Arun Shivalingam. "However, this kills them and brings into question whether the molecule really interacts with quadruplexes in a dynamic environment."
"We've shown that our molecule could be potentially used to verify in live cells and in real time whether potential quadruplex DNA binders are hitting their target," Vilar adds. "This could be a game-changer to accelerate research into these DNA structures."
Full details of the work appear in the journal Nature Communications; for more information, please visit http://dx.doi.org/10.1038/ncomms9178.
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