Quadruple Helix DNA

A team of scientists from the United Kingdom and Spain has demonstrated that a technique called fluorescence lifetime imaging microscopy in conjunction with a fluorescent probe can identify four-stranded ‘quadruple helix’ DNA structures within nuclei of live cells.

Fluorescence lifetime imaging microscopy map of nuclear DNA in live cells stained with the DAOTA-M2 probe; colors represent fluorescence lifetimes between 9 (red) and 13 (blue) nanoseconds. Scale bars – 20 µm. Image credit: Summers et al., doi: 10.1038/s41467-020-20414-7.

Guanine-rich sequences of DNA can fold into tetra-stranded helical assemblies known as G-quadruplexes.

These structures are implicated in a number of essential biological processes such as telomere maintenance, transcription, translation, and replication.

“A different DNA shape will have an enormous impact on all processes involving it — such as reading, copying, or expressing genetic information,” said co-lead author Dr. Ben Lewis, a researcher at Imperial College London and the MRC London Institute of Medical Sciences.

“Evidence has been mounting that G-quadruplexes play an important role in a wide variety of processes vital for life, and in a range of diseases, but the missing link has been imaging this structure directly in living cells.”

“G-quadruplexes are rare inside cells, meaning standard techniques for detecting such molecules have difficulty detecting them specifically.”

“It’s like finding a needle in a haystack, but the needle is also made of hay.”

To solve the problem, Dr. Lewis and colleagues used a chemical probe called DAOTA-M2, which fluoresces in the presence of G-quadruplexes, but instead of monitoring the brightness of fluorescence, they monitored how long this fluorescence lasts.

This signal does not depend on the concentration of the probe or of G-quadruplexes, meaning it can be used to unequivocally visualize these rare molecules.

“By applying this more sophisticated approach we can remove the difficulties which have prevented the development of reliable probes for this DNA structure,” said co-senior author Dr. Marina Kuimova, a researcher at Imperial College London.

The authors used their probes to study the interaction of G-quadruplexes with two helicase proteins — molecules that ‘unwind’ DNA structures.

They showed that if these helicase proteins were removed, more G-quadruplexes were present, showing that the helicases play a role in unwinding and thus breaking down G-quadruplexes.

“In the past we have had to rely on looking at indirect signs of the effect of these helicases, but now we take a look at them directly inside live cells,” said co-senior author Dr. Jean-Baptiste Vannier, a researcher at Imperial College London and the MRC London Institute of Medical Sciences.

The team also examined the ability of other molecules to interact with G-quadruplexes in living cells.

If a molecule introduced to a cell binds to this DNA structure, it will displace the DAOTA-M2 probe and reduce its lifetime; how long the fluorescence lasts.

This allows interactions to be studied inside the nucleus of living cells, and for more molecules, such as those which are not fluorescent and can’t be seen under the microscope, to be better understood.

“Many researchers have been interested in the potential of G-quadruplex binding molecules as potential drugs for diseases such as cancers,” said co-senior author Professor Ramon Vilar, a researcher at Imperial College London.

“Our method will help to progress our understanding of these potential new drugs.”

The team’s paper was published in the journal Nature Communications.

_____

P.A. Summers et al. 2021. Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy. Nat Commun 12, 162; doi: 10.1038/s41467-020-20414-7