For the first time, scientists have detected a DNA structure inside living human cells that looks more like a four-stranded knot than the elegant double helix we learned about in school.

The tangled shape, known as an i-motif, had been seen before in the lab, but few researchers expected it to occur in human cells.

The new work shows not only that i-motifs do indeed exist in human cells, but that they may be quite common.

"Our imaging suggests that this is a normal thing that happens," said Marcel Dinger, a molecular biologist at the Garvan Institute for Medical Research in Sydney, Australia, who oversaw the research. "It is very likely that genomes in all the cells of our bodies are forming i-motifs at some point in time."

A report on the find was published Monday in Nature Chemistry.

The study lends credence to the idea that these unusual DNA shapes may play an essential role in human biology, said Laurence Hurley, a professor of medical chemistry at the University of Arizona who was not involved with the work.

Perhaps i-motifs help the body control when genes are turned on to make proteins and when they are quiet, Hurley said. Whatever they do, they are sure to be important for chemical biology and molecular therapeutics, he added.

The DNA in our cells spends most of its time in the familiar double helix structure. But even in this stable shape, the molecule is constantly in flux.

When a piece of DNA is being replicated, the two strands are pulled apart and paired with new sequences that are assembled to match.

DNA molecules also separate when the instructions for a gene are being read by the cell. When the process is over, the strands zip back together.

The four-stranded i-motif occurs only in a relatively small region of a genome — sticking out like a bumpy knot in the smooth helical form.

An illustration of an i-motif embedded in a strand of DNA. (Mahdi Zeraati.)
 
To be clear, not just any piece of DNA can fold itself into the i-motif shape. There must be a specific sequence of letters that include several cytosines, which are written as Cs in the genetic code.

Back in the early 1990s, French scientists playing around in the laboratory discovered that a cytosine-rich region of a DNA strand could fold on top of itself, creating a four-stranded shape in which Cs paired with Cs instead of their usual partners, guanines, or Gs.

The researchers dubbed it an i-motif. The "i" stands for "intercalation," which is a chemistry term for a layered structure.

In lab experiments, it seemed that this DNA origami could occur only under acidic conditions that did not exist inside a cell.

"It was thought to be a weird idiosyncratic thing that the molecule can do, but not relevant for human biology," Dinger said.

But then other studies started to poke holes in that theory.

For example, researchers showed that an i-motif shape could form in an environment that was extremely crowded, even if it wasn't so acidic. The nucleus of a cell could certainly be crowded enough for this to occur.

Following a hunch, Dinger and his colleagues decided to see if they could find i-motifs inside living cells.

To do this, they worked with Daniel Christ, the head of antibody therapeutics at the Garvan Institute. After lots of trial and error, scientists in Christ's lab were able to develop an antibody that could search out i-motifs in the genome and bind to them.

The antibodies were tagged with a biological marker that glows when a fluorescent light shines on it. By looking at a strand of DNA under a special microscope, the researchers were able to see whether an i-motif occurred by looking for fluorescent dots. The more i-motifs there were, the more dots they would see.

I-motifs are what scientists call "dynamic" — they can fold and unfold depending on the acidity of their surroundings.

In addition, the sequences that code for i-motifs are generally found not within a gene itself. Instead, they're a little upstream, in a part of the genome known as the promoter region that determines whether or not a certain gene gets turned on.

These two facts suggest that i-motifs may be used as a type of switch that can regulate gene expression, said Randy Wadkins, a biochemist at the University of Mississippi in Oxford who was not involved with the new study.

It's possible that certain stressors can cause the acidity of the cell to change and prompt an i-motif to form. This, in turn, could trigger an over-expression or an under-expression of a nearby gene, Wadkins said.

"Think of it like a dial," he said. "But for now, we don't know whether that switch turns it up to 11 or turns it down to 0."

However, it is also possible that these i-motifs do nothing at all.

"The caveat for all of this is that these antibodies may have just trapped these oddball structures when they were forming and they don't have any significance," he said.

Indeed, scientists have long known about other shapes that DNA can fold into in the lab, including ones that resemble cruciforms and hair pins.

"DNA is a conformationally flexible molecule," Wadkins said. "But the question is, does this stuff have any biological relevance?"

For his part, Wadkins thinks it is likely that i-motifs do play a role in gene expression, but he said more work will be needed to say for certain.

And now that researchers know these strange four-stranded structures do frequently occur in human DNA, they are ready to find out.

"This opens up a whole new line of science," Dinger said.

Los Angeles Times

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