An Advanced Method of DNA Nanostructure Formation Developed

Figure 1: Uni-molecular magnetic tweezers orchestrating the DNA nanostructure formation

Professor Tae-Young Yoon’s research team from the Department of Physics at KAIST has developed a new method to form DNA nanostructures by using magnetic tweezers to observe and to induce the formation of the structure in real time.

Unlike traditional designs of "DNA origami" which relies on thermal or chemical annealing methods, the new technology utilizes a completely different dynamic in DNA folding. This allows the folding to be done within only ten minutes.

Developed in 2006, the "DNA origami" allows a long skeleton of DNA to be folded into an arbitrary structure by using small stapler DNA pieces. This has been a prominent method in DNA nanotechnology.
 

Figure 2: The evolution of DNA nanostructure formation using magnetic tweezers. The DNA nanostructure with a 21-nanometer size was formed in about eight minutes.

However, the traditional technology which adopts thermal processes could not control the DNA formation during the folding because every interaction among DNAs occurs simultaneously. Thus, the thermal processes, which take dozens of hours to complete, had to be repeated multiple times in order to find the optimal condition.

The research team designed a DNA folding using uni-molecular magnetic tweezers that applied force to a single DNA molecule while measuring the state of the DNA. Through this technology, they were able to induce the formation of DNA nanostructure and observe it at the same time.

During high temperature heat treatment, the first stage of conventional thermal processes, the internal structure of the long skeleton DNA untangles. To induce such state, after attaching one side of the skeleton DNA to the surface of glass and the other side to a magnetic material, the team unfolded the internal structure of the DNA by pulling the two sides apart with magnetic force.

Unlike the conventional thermal processes, this method lets the stapler DNA swiftly adhere to the skeleton DNA within a minute because the sites are revealed at room temperature.

After the stapler pieces connected to the skeleton, the team removed the magnetic force. Next, the structure folded through self-assembly as the stapler DNAs stuck to different sites on the skeleton DNA.

Professor Yoon said, “With the existing thermal methods, we could not differentiate the reactions of the DNA because the response of each DNA pieces mutually interacted with each other.” He added that “Using the magnetic tweezers, we were able to sort the process of DNA nanostructure formation into a series of reactions of DNA molecules that are well known, and shorten the time taken for formation in only ten minutes.”

He commented, “This nanostructure formation method will enable us to create more intricate and desirable DNA nanostructures by programming the folding of DNA origami structures.”

Conducted by Dr. Woori Bae under the guidance of Professor Yoon, the research findings were published online in the December 4th issue of Nature Communications.


Source: KAIST

First steps in formation of pancreatic cancer identified

Shown is a region of a pancreas with preneoplastic lesions. Red labeling indicates macrophages, green labeling indicates pancreatic acinar cells that dedifferentiate, and grey labeling indicates further progressed pancreatic lesions. Credit: Image courtesy of Mayo Clinic

Researchers at Mayo Clinic's campus in Jacksonville say they have identified first steps in the origin of pancreatic cancer and that their findings suggest preventive strategies to explore.

In an online issue of Cancer Discovery, the scientists described the molecular steps necessary for acinar cells in the pancreas -- the cells that release digestive enzymes -- to become precancerous lesions. Some of these lesions can then morph into cancer.

"Pancreatic cancer develops from these lesions, so if we understand how these lesions come about, we may be able to stop the cancer train altogether," says the study's lead investigator, Peter Storz, Ph.D., a cancer biologist.

The need for new treatment and prevention strategies is pressing, Dr. Storz says. Pancreatic cancer is one of the most aggressive human cancers -- symptoms do not occur until the cancer is well advanced. One-year survival after diagnosis is only 20 percent. It is the fourth leading cause of cancer death in this country.

The scientists studied pancreatic cells with Kras genetic mutations. Kras produces a protein 

that regulates cell division, and the gene is often mutated in many cancers. More than 95 percent of pancreatic cancer cases have a Kras mutation.

The researchers detailed the steps that led acinar cells with Kras mutations to transform into duct-like cells with stem cell-like properties. Stem cells, which can divide at will, are also often implicated in cancer.

They found that Kras proteins in the acinar cells induce the expression of a molecule, ICAM-1, which attracts macrophages, a specific kind of immune cells. These inflammatory macrophages release a variety of proteins, including some that loosen the structure of the cells, allowing acinar cells to morph into different types of cells. These steps produced the precancerous pancreatic lesions.

"We show a direct link between Kras mutations and the inflammatory environment that drive the initiation of pancreatic cancer," Dr. Storz says.

But the process can be halted in laboratory mice, he adds. "We could do this two ways -- by depleting the macrophages or by treating the transformed cells with a blocking antibody that shuts down ICAM-1," says Dr. Storz. "Doing either one reduced the number of precancerous lesions."

Dr. Storz noted that a neutralizing antibody that blocks ICAM-1has already been developed. It is being tested for a wide variety of disorders, including stroke and rheumatoid arthritis.

"Understanding the crosstalk between acinar cells with Kras mutations and the microenvironment of those cells is key to developing targeted strategies to prevent and treat this cancer," he says.

Source: Mayo Clinic

Scientists discover new way protein degradation is regulated

Chamber of doom. Rockefeller scientists have identified a new way that proteins are degraded in the proteasome (green). They found that the enzyme tankyrase regulates proteasome activity by promoting the assembly of proteasome subunits into the active complex called 26S. Credit: Image by Sigi Benjamin-Hong, Strang Laboratory of Apoptosis and Cancer Biology

Proteins, unlike diamonds, aren't forever. And when they wear out, they need to be degraded in the cell back into amino acids, where they will be recycled into new proteins. Researchers at Rockefeller University and the Howard Hughes Medical Institute have identified a new way that the cell's protein recycler, the proteasome, takes care of unwanted and potentially toxic proteins, a finding that has implications for treating muscle wasting, neurodegeneration and cancer.

The consensus among scientists has been that the proteasome is constantly active, chewing up proteins that have exceeded their shelf life. A mounting body of evidence now suggests that the proteasome is dynamically regulated, ramping up its activity when the cell is challenged with especially heavy protein turnover. The researchers, postdoctoral associate Park F. Cho-Park and Hermann Steller, head of the Strang Laboratory of Apoptosis and Cancer Biology at Rockefeller, have shown that an enzyme called tankyrase regulates the proteasome's activity. In addition, Cho-Park and Steller demonstrate that a small molecule called XAV939, originally identified by scientists at Novartis who developed it as therapeutic for colon cancer, inhibits tankyrase and blocks the proteasome's activity. The research is reported in today's issue of the journal Cell.

"Our findings have tremendous implications for the clinic since it gives a new meaning to an existing class of small-molecule compound," says Steller, Strang Professor at Rockefeller and an investigator at HHMI. "In particular, our work suggests that tankyrase inhibitors may be clinically useful for treating multiple myeloma."

Tankyrase was originally identified in the late 1990s by Rockefeller's Titia de Lange and her colleagues in the Laboratory for Cell Biology and Genetics, who showed that it plays a role in elongating telomeres, structures that cap and protect the ends of chromosomes. In a series of experiments in fly and human cells, Cho-Park and Steller discovered that tankyrase uses a process called ADP-ribosylation to modify PI31, a key factor that regulates the activity and assembly of proteasome subunits into the active complex called 26S. By promoting the assembly of more 26S particles, cells under stress can boost their ability to break down and dispose of unwanted proteins.

The proteasome is currently a target for developing cancer therapeutics. The FDA has approved Velcade, a proteasome inhibitor, for the treatment of multiple myeloma and mantle cell lymphoma. However, patients on Velcade can experience peripheral neuropathy or become resistant to the drug.

Multiple myeloma cells need increased proteasome activity to survive. Preliminary data from Cho-Park and Steller show that XAV939 can block the growth of multiple myeloma cells by inhibiting the assembly of additional proteasomes without affecting the basal level of proteasomes in the cell. This selective targeting may mean fewer side effects for patients. 

"Drugs, such as XAV939, that inhibit the proteasome through other mechanisms than Velcade may have significant clinical value," says Steller.

The findings by Cho-Park and Steller also link, for the first time, metabolism and regulation of the proteasome. Sometimes the proteasome digests too much protein, which can lead to loss of muscle, says Steller.

"This discovery reveals fundamental insights into protein degradation, a process important for normal cell biology, and a key factor in disorders such as muscle wasting and neurodegeneration," said Stefan Maas of the National Institutes of Health's National Institute of General Medical Sciences, which partly supported the study. "Intriguingly, the findings also enlighten ongoing research on cancer therapies, exemplifying the impact of basic research on drug development."

Source: Rockefeller University

Source of most cases of invasive bladder cancer identified

Philip Beachy and his team found a single type of cell in mice that gives rise to invasive bladder cancers. Credit: Steve Fisch

A single type of cell in the lining of the bladder is responsible for most cases of invasive bladder cancer, according to researchers at the Stanford University School of Medicine.

Their study, conducted in mice, is the first to pinpoint the normal cell type that can give rise to invasive bladder cancers. It's also the first to show that most bladder cancers and their associated precancerous lesions arise from just one cell, and explains why many human bladder cancers recur after therapy.

"We've learned that, at an intermediate stage during cancer progression, a single cancer stem cell and its progeny can quickly and completely replace the entire bladder lining," said Philip Beachy, PhD, professor of biochemistry and of developmental biology. "All of these cells have already taken several steps along the path to becoming an aggressive tumor. Thus, even when invasive carcinomas are successfully removed through surgery, this corrupted lining remains in place and has a high probability of progression."

Although the cancer stem cells, and the precancerous lesions they form in the bladder lining, universally express an important signaling protein called sonic hedgehog, the cells of subsequent invasive cancers invariably do not -- a critical switch that appears vital for invasion and metastasis. This switch may explain certain confusing aspects of previous studies on the cellular origins of bladder cancer in humans. It also pinpoints a possible weak link in cancer progression that could be targeted by therapies.

"This could be a game changer in terms of therapeutic and diagnostic approaches," said Michael Hsieh, MD, PhD, assistant professor of urology and a co-author of the study. "Until now, it's not been clear whether bladder cancers arise as the result of cancerous mutations in many cells in the bladder lining as the result of ongoing exposure to toxins excreted in the urine, or if it's due instead to a defect in one cell or cell type. If we can better understand how bladder cancers begin and progress, we may be able to target the cancer stem cell, or to find molecular markers to enable earlier diagnosis and disease monitoring."

Beachy is the senior author of the study, which will be published online April 20 in Nature Cell Biology. He is the Ernest and Amelia Gallo Professor in the School of Medicine and a member of the Stanford Cancer Institute and the Stanford Institute for Stem Cell Biology and Regenerative Medicine. He is also a Howard Hughes Medical Institute investigator. Kunyoo Shin, PhD, an instructor at the institute, is the lead author.

Bladder cancer is the fourth most common cancer in men and the ninth most common in women. Smoking is a significant risk factor. There are two main types of the disease: one that invades the muscle around the bladder and metastasizes to other organs, and another that remains confined to the bladder lining. Unlike the more-treatable, noninvasive cancer -- which comprises about 70 percent of bladder cancers -- the invasive form is largely incurable. It is expensive and difficult to treat, and the high likelihood of recurrence requires ongoing monitoring after treatment.

In 2011, Shin and Beachy and their colleagues identified a cell type in the bladder that is capable of completely replacing the lining of the organ after infection or damage. The fact that it could give rise to multiple cell types (even forming small, multilayered, bladder-like spheres when cultured in vitro), and also self-renew, showed that it was a bladder stem cell. 

They found that the cell, which came from the basal layer of the bladder epithelium, used a protein called sonic hedgehog to "talk" to other cells in the bladder and stimulate proliferation and specialization into other cell types. (Beachy identified the first hedgehog protein in fruit flies in 1992; the hedgehog signaling pathway has since been shown to play a vital role in embryonic development and in many types of cancers.)

Many animal models of cancer rely on prior knowledge or hunches as to what genes or cell types are involved. Researchers may genetically alter an animal, or a certain cell type, to induce the overexpression of a gene known to be involved in tumorigenesis, for example, or block the expression of a gene that inhibits cancer development.

Although prior work suggested that basal cells may play a role in bladder cancer, the researchers chose an unbiased approach when developing their mouse model that more closely mimicked what happens in humans: They put a chemical compound called N-butyl-N-4-hydroxybutyl nitrosamine, or BBN, in the mice's drinking water and watched the animals over a period of months.

Nitrosamines are carcinogens found in cigarette smoke; BBN is a form of the chemical that is specifically activated in the bladder. After four months, many of the animals had developed precancerous lesions, or carcinomas in situ, in their bladders that very closely resemble those seen in human patients. By six months, all of the animals had developed invasive bladder cancers.

With their model in place, the researchers then conducted two main experiments in the mice: In the first experiment, they looked to see what would happen in animals exposed to BBN when the sonic-hedgehog-expressing cells were marked with a distinctive fluorescent color. In the second, they used genetic techniques to selectively kill those same cells in animals prior to exposure with BBN.

In the first case, they saw something startling: After just a few months of BBN exposure, nearly the entire lining of the bladder was labeled with the fluorescent green marker that indicated the cells had arisen from the sonic-hedgehog-expressing basal stem cells. When transplanted into other mice, those labeled cells were able to give rise to bladder cancers, but cells not expressing sonic hedgehog did not.

In the second case, no tumors grew in the animals in which the stem cells had been selectively killed -- although the bladder architecture became severely compromised in the absence of stem cells to regenerate cells lost during the normal course of bladder function.

"So now we have two lines of evidence indicating that the bladder stem cells are solely responsible for tumorigenesis," Shin said. "When we mark the bladder stem cells, the tumors are also marked. When we remove, or ablate, the stem cells, no tumors arise after BBN treatment."

Next the researchers tackled the question of whether bladder cancers arise as the result of genetic changes to one or more of these bladder stem cells. To do so, they used a genetically engineered mouse with cells that fluoresce green, but which can be triggered to randomly fluoresce one of three other colors: red, blue or yellow. Known as a "rainbow mouse," the animal allows researchers to more precisely determine the origin of groups of cells. If all cells in a tumor are red, for example, it is much more likely that they originated from a single cell.

"After four months of BBN treatment," Beachy said, "we'd most often see just one color dominating the entire epithelium. This clearly indicates that a single cell has taken over the lining of the entire bladder, elbowing out its neighbors in a way that's not been seen in other organs."

Further studies showed that, surprisingly, none of the cells in the most advanced, invasive carcinomas in the BNN-treated animals expressed sonic hedgehog -- despite the fact that only sonic-hedgehog-expressing cells are able to give rise to the earlier stages of bladder cancer. One obvious implication of the lack of sonic hedgehog expression in these cells is that the hedgehog pathway somehow inhibits steps required for tissue invasion or metastasis.

"We know that the hedgehog pathway is widely used throughout the animal kingdom to tightly regulate cellular and tissue differentiation," Hsieh said. "So its loss could make sense in this context because cancer is essentially a loss of normal regulation."

"One really important lesson from this study," Beachy said, "is the idea that, by the time you get to a full-blown tumor, the properties of the cells in that tumor may have changed quite significantly from the cell type that gives rise to the tumors. This can complicate understanding how human tumors arise, because even if you identify the tumor-propagating cells within a mature tumor, conclusions about the origins of a cancer based on properties of these cells may be inaccurate."

Source: Stanford University Medical Center

Charged graphene gives DNA a stage to perform molecular gymnastics

DNA interacts with charged graphene and contorts into sequence-specific shapes when the charge is changed. Credit: Photo courtesy Alek Aksimentiev

When Illinois researchers set out to investigate a method to control how DNA moves through a tiny sequencing device, they did not know they were about to witness a display of molecular gymnastics.

Fast, accurate and affordable DNA sequencing is the first step toward personalized medicine. Threading a DNA molecule through a tiny hole, called a nanopore, in a sheet of graphene allows researchers to read the DNA sequence; however, they have limited control over how fast the DNA moves through the pore. In a new study published in the journal Nature Communications, University of Illinois physics professor Aleksei Aksimentiev and graduate student Manish Shankla applied an electric charge to the graphene sheet, hoping that the DNA would react to the charge in a way that would let them control its movement down to each individual link, or nucleotide, in the DNA chain.

"Ideally, you would want to step the DNA through the nanopore one nucleotide at a time," said Aksimentiev. "Take a measurement and then have another nucleotide in the sensing hole. That's the goal, and it hasn't been realized yet. We show that, to some degree, we can control the process by charging the graphene."

The researchers found that a positive charge in the graphene speeds up DNA movement through the nanopore, while a negative charge stops the DNA in its tracks. However, as they watched, the DNA seemed to dance across the graphene surface, pirouetting into shapes they had never seen, specific to the sequence of the DNA nucleotides.

"It reminds me of Swan Lake," Aksimentiev said. "It's very acrobatic. We were very surprised by the variety of DNA conformations that we can observe at the surface of graphene when we charge it. There is one sequence that starts out laying down on the surface, and when we change the charge, they all tilt on the side like they are doing a one-armed push-up. Then we also have nucleotides that would lay back, or go up like a ballerina en pointe."

Aksimentiev hypothesizes that the conformations are so different and so specific to the sequence because each nucleotide has a slightly different distribution of electrons, the negatively charged parts of the atoms. There is even a visible difference when a nucleotide is methylated, a tiny chemical change that can turn a gene on or off.

By switching the charge in the graphene, the researchers can control not only the DNA's motion through the pore, but also the shape the DNA contorts into.

"Because it's reversible, we can force it to adopt one conformation and then force it to go back. That's why we call it gymnastics," Aksimentiev said.

The researchers extensively used the Blue Waters supercomputer at the National Center for Supercomputing Applications, housed at the University of Illinois. They mapped each individual atom in the complex DNA molecule and ran numerous simulations of many different DNA sequences. Supercomputing power was essential to carrying out the work, Aksimentiev said.

"This is a really computationally intensive project," he said. "Having access to Blue Waters was essential because with the sheer number of simulations, we would not have been able to finish them. It would have taken too long."

The next step is to combine a charged nanopore setup with a sensor to build a DNA sequencing device that would incorporate both motion control and nucleotide recognition. The researchers also hope to explore the unexpected conformational changes for insights into epigenetics, the field that studies how genes are expressed and moderated.

"DNA is much more complicated than just a double helix. It's a complex molecule that has many properties, and we are still uncovering them," Aksimentiev said.

Video animation of DNA dancing as the graphene charge changes:


Source: University of Illinois at Urbana-Champaign