Scientists Find Folded G-Quadruplex DNA Structures in Human Blood Plasma

If you believe you know what DNA looks like, your blood may hold an unexpected surprise.

DNA is usually pictured as the iconic double helix. Yet genetic material does not invariably keep this well-known shape: some fragments can curl into dense, four-stranded forms.

Scientists have observed these unusual structures within cells before. They now report detecting them in their folded state in human blood plasma.

Why DNA enters the bloodstream

Why is there DNA in blood in the first place?

When cells die across the body, they shed tiny DNA fragments. Like scraps from a ripped-up note, these enter the bloodstream carrying hints about processes occurring inside the body.

Researchers have traditionally focused on deciphering that message: the genetic letters it contains, the size of the fragments and their location in the genome. However, a new preprint study indicates that the way this message is folded may also be important.

The researchers found four-stranded formations known as G-quadruplexes, or G4s, in genetic material collected directly from human plasma.

"Our study provides, to our knowledge, the first direct biochemical evidence that folded G-quadruplex structures are present in nucleic acids captured directly from human plasma," University of Cologne molecular biologist Robert Hänsel-Hertsch told ScienceAlert.

A G4 forms when four instances of guanine, a genetic letter, come together to make a minute square. Multiple squares can then pile up, producing a compact arrangement unlike the familiar double helix.

Detecting folded G-quadruplexes in human plasma

The puzzle started with the discovery of unusual pieces called ultrashort cell-free DNA. At around 50 genetic letters in length, these single strands are sufficiently small to pass through the traps used in conventional DNA-capture techniques.

Certain fragments had enough guanine to create G4s, while computer analysis suggested the same conclusion.

However, locating a strand capable of making a knot is different from finding that strand already tied. The researchers needed to capture the structures in human blood while they remained folded.

This posed a greater challenge than it might seem. The aggressive methods used to extract DNA from plasma may unravel fragile structures or let molecules refold during the experiment. Consequently, if a G4 was found at the end of the process, the team could not tell whether it had been present in the bloodstream or formed in the laboratory.

The team therefore used a more delicate approach. They collected genetic material directly from plasma, avoiding heating and extraction procedures that could alter its form.

They next deployed two separate detection tools: an antibody engineered to identify G4s, and a small molecule that glows upon binding to one.

Both methods generated a signal.

The researchers also captured a corresponding genetic sequence unable to fold, but it generated no signal. They further checked their finding by having the two G4-detection agents compete for the same structures. Each weakened the other's signal, suggesting both were identifying folded G4s instead of attaching at random to the captured material.

"Structure is a layer of information, and potentially of biology, that sequencing alone cannot see," Hänsel-Hertsch said.

What G4 structures could reveal about health

Yet the finding raised a further question.

Although the study began with ultrashort DNA, the experiment could not determine whether the detected G4s consisted of DNA or RNA. RNA is able to adopt the same four-stranded form, and the detection tools recognise the shape rather than the underlying molecule.

Hänsel-Hertsch called this "a real limitation".

A forthcoming step will involve treating the collected material with enzymes that destroy either DNA or RNA, followed by further testing. Sequencing the folded molecules could then establish their identity and where they originated in the genome.

Scientists also remain unsure which tissues released these molecules, or whether the structures folded within cells or only once they entered the bloodstream.

What, then, could they reveal about health?

G4s commonly appear in genomic regions that help regulate genes, and elevated G4 levels have been seen in several tumour types. This suggests that blood-circulating G4s might eventually offer information about cancer that cannot be obtained by reading DNA sequences alone.

This is not, however, a new cancer test.

The research aimed to demonstrate that folded G4s exist in plasma, rather than compare people with cancer with healthy participants. Any diagnostic use would first require researchers to identify the molecules and measure them in carefully characterised groups of patients and healthy volunteers.

Only after that will they be able to determine whether disease alters the quantity, source or pattern of these folded structures in a meaningful way.

For the moment, the study presents a more basic - and surprising - idea. The genetic debris carried in our blood may be more than a sequence of letters waiting to be read. At times, the way that paper is folded could itself form part of the message.

Scientists are only beginning to learn how to interpret it.

The preprint study is available on bioRxiv.

This article was fact-checked by Fiona MacDonald and edited by Fiona MacDonald. Although we take pride in our process, we are only human. If you notice an error, please let us know.