Formation of tracked quadruple helix DNA in living human cells for the first time

DNA generally forms the classic double helix shape discovered in 1953: two strands coiled together. Several other structures have been formed in test tubes, but this does not necessarily mean that they form within living cells.

Quadruple helix structures, called DNA G-quadruplexes (G4s), have previously been detected in cells. However, the technique used required killing the cells or using high concentrations of chemical probes to visualize the formation of G4, so its actual presence within living cells under normal conditions has not been tracked, until now.

A research team from Cambridge University, Imperial College London and the University of Leeds has invented a fluorescent marker that can bind to G4 in living human cells, allowing them to see for the first time how the structure is formed. and what role does it play. cells.

The study is published today in Chemistry of nature.

Rethink the biology of DNA.

One of the principal investigators, Dr. Marco Di Antonio, who began work at Cambridge University in Professor Sir Shankar Balasubramanian’s laboratory and now heads a research team in the Department of Chemistry at Imperial, said: “For the first time Once, we have been able to demonstrate that the DNA of the quadruple helix exists in our cells as a stable structure created by normal cellular processes. 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 such as cancer.

“Now we can track G4 in real time in cells, we can ask directly what its biological role is. We know it appears to be more prevalent in cancer cells, and now we can investigate what role it is playing and potentially how to block it, potentially devising new therapies.” .

The team believes that G4 forms in DNA to temporarily keep it open and facilitate processes such as transcription, where DNA instructions are read and proteins are made. This is a form of ‘gene expression’, where part of the genetic code in DNA is activated.

G4s appear to be most often associated with genes involved in cancer, and are detected in large numbers within cancer cells. With the ability to image a single G4 at a time, the team says they could trace their role within specific genes and how they are expressed in cancer. This fundamental knowledge could reveal new drug targets that disrupt the process.

Natural formation

The team’s advancement in the ability to image individual G4s came with a rethink of the mechanisms generally used to investigate cell function. Previously, the team had used antibodies and molecules that could find and bind to G4, but these required very high concentrations of the ‘probe’ molecule. This meant that the molecules in the probe could be altering DNA and actually causing it to form G4, rather than detecting its natural formation.

Dr. Aleks Ponjavic, now an academic at the University of Leeds Schools of Physics and Astronomy and Food Science and Nutrition, jointly led the research in Professor Sir David Klenerman’s laboratory and developed the method of visualizing the new fluorescent marker with microscopy. .

He said: “Scientists need special probes to see the molecules inside living cells, however these probes can sometimes interact with the object we are trying to see. By using single molecule microscopy, we can observe probes at concentrations 1000 times lower than previously used. In this case, our probe binds to G4 for only milliseconds without affecting its stability, allowing us to study the behavior of G4 in its natural environment without external influence. “

For the new probe, the team used a very ‘bright’ fluorescent molecule in small amounts that was designed to adhere to G4 very easily. The small amounts meant that they could not expect to image every G4 in a cell, but instead could identify and track individual G4s, allowing them to understand their fundamental biological role without disturbing their prevalence and overall stability in the cell.

The team was able to demonstrate that the G4 appear to form and dissipate very quickly, suggesting that they only form to perform a certain function, and that potentially if they last too long they can be toxic to normal cellular processes.