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Four-Stranded DNA

Researchers Observe Formation of Four-Stranded DNA in Living Human Cells

DNA usually forms the classic double helix shape — two strands wound around each other. Several other structures have been formed in the lab, but this does not necessarily mean they form within living cells. Quadruple helix structures, called DNA G-quadruplexes, have previously been detected in cells. However, the technique used required either killing the cells or using high concentrations of chemical probes to visualize their formation, so their actual presence within living cells under normal conditions has not been tracked. Now, a research team led by University of Cambridge scientists has invented a fluorescent marker that is able to attach to DNA G-quadruplexes in living human cells.

Illustration of quadruple helix DNA (green) forming. Image credit: Ella Maru Studio.

Illustration of quadruple helix DNA (green) forming. Image credit: Ella Maru Studio.

“For the first time, we have been able to prove the quadruple helix DNA exists in our cells as a stable structure created by normal cellular processes,” said first author Dr. Marco Di Antonio, a researcher in the Chemistry Department at Imperial College London.

“This forces us to rethink the biology of DNA. It is a new area of fundamental biology, and could open up new avenues in diagnosis and therapy of diseases like cancer.”

“Now we can track DNA G-quadruplexes in real time in cells we can ask directly what their biological role is. We know it appears to be more prevalent in cancer cells and now we can probe what role it is playing and potentially how to block it, potentially devising new therapies.”

Dr. Di Antonio and colleagues think G-quadruplexes form in DNA in order to temporarily hold it open and facilitate processes like transcription, where the DNA instructions are read and proteins are made. This is a form of gene expression, where part of the genetic code in the DNA is activated.

DNA G-quadruplexes appear to be associated more often with genes involved in cancer, and are detected in larger numbers within cancer cells.

“With the ability to now image a single DNA G-quadruplex at a time, we could track their role within specific genes and how these express in cancer,” the scientists said.

“This fundamental knowledge could reveal new targets for drugs that interrupt the process.”

Microscopy image of the fluorescent quadruple helix DNA. Image credit: Di Antonio et al, doi: 10.1038/s41557-020-0506-4.

Microscopy image of the fluorescent quadruple helix DNA. Image credit: Di Antonio et al, doi: 10.1038/s41557-020-0506-4.

The researchers used a very bright fluorescent molecule in small amounts that was designed to stick to the DNA G-quadruplexes very easily.

The small amounts meant they couldn’t hope to image every DNA G-quadruplex in a cell, but could instead identify and track single DNA G-quadruplexes, allowing them to understand their fundamental biological role without perturbing their overall prevalence and stability in the cell.

The authors were able to show that DNA G-quadruplexes appear to form and dissipate very quickly, suggesting they only form to perform a certain function, and that potentially if they lasted too long they could be toxic to normal cell processes.

“Scientists need special probes to see molecules within living cells, however these probes can sometimes interact with the object we are trying to see,” said co-author Dr. Aleks Ponjavic, a researcher in the Schools of Physics Astronomy and Food Science and Nutrition at the University of Leeds.

“By using single-molecule microscopy, we can observe probes at 1,000-fold lower concentrations than previously used.”

“In this case, our probe binds to the DNA G-quadruplexes for just milliseconds without affecting its stability, which allows us to study the behavior of DNA G-quadruplexes in their natural environment without external influence.”

The results were published in the journal Nature Chemistry.

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M. Di Antonio et al. Single-molecule visualization of DNA G-quadruplex formation in live cells. Nat. Chem, published online July 20, 2020; doi: 10.1038/s41557-020-0506-4

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