Chemical cages: New technique advances synthetic biology


February 10, 2016

Living systems rely on a dizzying variety of chemical reactions essential to development and survival. Most of these involve a specialized class of protein molecules — the enzymes.

In a new study, Hao Yan, director of the Center for Molecular Design and Biomimetics at Arizona State University’s Biodesign Institute, presents a clever means of localizing and confining enzymes and the substrate molecules they bind with, speeding up reactions essential for life processes. Enzyme nanocages illustration Download Full Image

The research, which appears in the current issue of the journal Nature Communications, could have far-reaching applications in fields ranging from improving industrial efficiencies to pioneering new medical diagnostics, guiding targeted drug delivery and producing smart materials. The work also promises to shed new light on particulars of cellular organization and metabolism.

The technique involves the design of specialized, nanometer-scale cages, which self-assemble from lengths of DNA. The cages hold enzyme and substrate in close proximity, considerably accelerating the rate of reactions and shielding them from degradation. 

“We have been designing programmable DNA nanostructures with increasing complexity for many years, and it is now time to ask what can we do with these structures,” said Yan, who is also a professor in ASU’s School of Molecular Sciences in the College of Liberal Arts and Sciences. “There are numerous other applications from this emerging technology. Through our interdisciplinary collaborative effort, we here describe the use of designer DNA nanocages to compartmentalize enzymatic reactions in a confined environment. Drawing inspiration from nature, we have uncovered interesting properties, some unexpected.”

Zhao Zhao, a researcher in the Center for Molecular Design and Biomimetics, was the lead author of the paper, which was co-authored with researchers from ASU's Department of Chemistry, Rutgers and the University of Michigan.

Enzyme world

As chemical activators for virtually every reaction in the body, enzymes are key participants in the normal activity of cells, tissues, fluids and organs. Hundreds of thousands of metabolic enzymes are present in the human body, involved in diverse activities including DNA copying and repair and the transformation of glucose into useable energy. Elsewhere, some 22 digestive enzymes break down carbohydrates (amylases), fats (lipases ) and sugars (disaccharides), while so-called protease enzymes digest proteins.

Enzymes tend to be highly specific, not only in the useful functions they perform, but the precise substrates with which they will work. Substrate molecules of exactly the right size and shape bind with their appropriate enzymes as the correct key fits into the ridges and grooves of a lock.

Substrates latch onto enzyme molecules at a particular region known as the active site. Once enzyme and substrate have combined, a chemical product is formed and then released, returning the enzyme to its original configuration where it is ready to operate on a new molecule of substrate.

In order for such reactions to take place in an efficient manner, nature has devised methods of compartmentalization, forming natural reactor sites where enzyme-substrate reactions unfold. The cell itself is such a compartment, as are various membrane-bound organelles found in eukaryotes (cells containing a nucleus), including mitochondria, lysosomes and peroxisomes.

Compartmentalization of reactants helps to overcome a variety of challenges, bringing binding chemicals into cozy proximity, isolating enzyme-substrate complexes from competing reaction chemicals, improving the yield of product molecules produced and reducing the toxicity various intermediary chemicals can sometimes cause.

In order to induce or catalyze chemical reactions for a variety of purposes, synthetic biologists have copied a page from Nature’s recipe book, designing artificial compartments fabricated from proteins, lipids or the nucleic acids found in DNA (as in the current study).

Close encounters

Yan and his colleagues designed their synthetic reactors to house enzymes and their substrates, allowing chemical conversions to take place in a controlled environment. Each minute structure, measuring just 54 nanometers across, is something like a Faberge egg whose separate halves fit together to encapsulate their chemical contents. (A nanometer is one-billionth of a meter or roughly 80,000 times smaller than the width of a human hair.)

Using the base pairing properties of DNA’s four nucleotides — labeled A, T, C and G — allows nanoscale architects like Yan to construct myriad forms in two- and three-dimensions. In the new study, DNA nanocages were used to encapsulate metabolic enzymes with high assembly yield and fine-tuned control over reactants and products.

The construction of the nanocages takes place in two steps. First, individual enzymes are attached into open half-cage structures. Then, the half-cages are fitted together into a full, closed nanocage. To create the half-cages, a technique known as DNA origami is used. Lengths of viral DNA are prepared to self-assemble into a honeycomb lattice, with A nucleotides pairing with C and T with G.  

The open-sided half-cages of the DNA nanocages allow the access of large protein molecules into the nanocage’s internal cavity. The two half-cages are fitted together with the aid of short bridge DNA strands that bind with complementary DNA sequences extending from the edges of either half-cage (see animation below). The small gaps on each of the top and bottom surfaces of the DNA nanocage allow the diffusion of small molecules across the DNA walls.

Two-step nanocage: Individual enzymes (orange and green) are first attached to half-cage structures. Half-cages are then assembled into full cages, where reactants are brought into close proximity. Animation by Jason Drees for the Biodesign Institute at ASU

Probing the nanoscale

To examine the resulting structures Transmission Electron Microscopy was used, along with gel electrophoresis and single-molecule fluorescence experiments, which demonstrated that close to 100 percent of the DNA segments properly formed half-cage structures and more than 90 percent formed full cages. 

The study examined six different enzymes, ranging in size from the smallest, which measured ~44kD (kilodaltons) to the largest, ~ 450 kD. All six enzymes were successfully encapsulated in nanocages, though the yields varied according to enzyme size. The largest enzyme examined, known as β-galactosidase, showed the lowest yield of 64 percent.

Next, the activity of enzyme-substrate pairs was evaluated. In addition to bringing the enzyme-substrate pair into closer binding proximity, encapsulation in the nanocage is also believed to facilitate activity through the unique electrical charge density conditions within the nanocage.

Subsequent experiments demonstrated that most of the effect on enzyme-substrate activity in nanocages is due to the unique charge environment within nanocages, rather than enzyme-substrate proximity. The authors suggest that encapsulated enzymes exhibit higher activity within densely packed DNA cages as a result of the highly ordered, hydrogen-bonded water environment surrounding them.

An evaluation of enzyme activity showed a 4- to 10-fold increase for enzymes encapsulated in nanocages, compared with the activity of free enzymes. Enzyme turnover rate — defined as the maximum number of chemical conversions of substrate molecules per second — was inversely correlated with the size of encapsulated enzymes, with the smallest enzyme yielding the highest turnover.

Future cages

The DNA cages demonstrated their resiliency during the experiments, retaining their structural form throughout the enzymatic reactions. They also protected encapsulated enzymes from deactivation due to digestive chemicals, while permitting the uninterrupted diffusion of small-molecule substrates and reaction products through the nanopores of the DNA cage.

Encapsulation in nanocages was shown to increase the fraction of active enzyme molecules and their individual turnover numbers. The method thus provides a new molecular tool to modify the local environment surrounding enzymes and their substrates, opening the door to new applications in smart materials and biomedical applications. Among the latter are futuristic, programmable cages that could be used as nanoscale delivery mechanisms for a wide range of therapeutic agents.

 

Richard Harth

Science writer, Biodesign Institute at ASU

480-727-0378

 
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The wild chimps of Kibale

ASU professor continues to find surprises in studies in Uganda chimp project.
Less than 300,000 chimps in the wild; those numbers are expected to drop.
February 10, 2016

ASU primatologist continues to find surprises in a chimpanzee stronghold in Uganda

Kevin Langergraber has been studying chimpanzees in the wild for 15 years.

Before he became a professor and had to teach eight months out of the year, he’d spend six months to a year at a time in the field, 12 hours a day, seven days a week.

“The most surprising thing about the job is that you can still be surprised by the job,” said Langergraber, a primatologist and assistant professor in the School of Human Evolution and Social ChangeThe School of Human Evolution and Social Change is a unit of the College of Liberal Arts and Sciences. at Arizona State University.

“After a while you’d think, ‘OK — I’ve spent thousands of hours watching these guys. What is going to surprise me when I wake up and spend my 12-hour day in the forest?’ ” he said. “Despite all this time I’m still shocked when I go back and the chimps show me something I hadn’t seen before.”

Langergraber, who has been at ASU for a year and a half, co-directs the Ngogo Chimpanzee Project in Kibale National Park, Uganda, home to the largest wild chimp community in the world. About 200 individuals live in the 35-square-kilometer preserve in the middle of the 800-square-kilometer national park. The community has been studied since 1995.

An adult chimp.

Django, an adult male
chimp in the Ngogo
Chimpanzee Project
in Kibale National
Park, Uganda.
Top: Penelope, an
adult chimp, with
her female infant,
Kozena.

Photos by Kevin
Langergraber

One big surprise came last summer. In the beginning of the season, food was lean. When food supplies are down, chimps are hard to find because they split into tiny groups to forage. But food availability crept up, to the point where three of their favorite fruits ripened at the same time.

“They basically had more food than they knew what to do with at this point,” Langergraber said. “It was falling off the trees and sitting on the ground uneaten.”

About 30 adult females with 60 kids congregated. “There were these huge parties, these huge hordes of chimpanzees, which was very unusual, especially for females, which are more solitary than male chimpanzees,” he said. “They began exhibiting really male-like behavior.”

Chimps hunt monkeys, but it’s almost always done by males, in groups. Females do hunt, but much less than males.

“(The females) started going on this little mini-hunting binge, where they were hunting almost every day over the course of a few weeks,” Langergraber said. “That is something I’d never expected to see in my life. … I’ve been there for 15 years, and this is something out of the blue I think will be important for learning about why in general is it males hunt more than females and questions like that.”

Chimpanzees are classified as endangered by the International Union for the Conservation of Nature. They’re endangered throughout their range across equatorial Africa. The IUCN estimates there are less than 300,000 chimps in the wild, according to a rough estimate made in 2003. Those numbers are expected to drop.

“Due to high levels of exploitation, loss of habitat and habitat quality due to expanding human activities, this species is estimated to have experienced a significant population reduction in the past 20 to 30 years, and it is suspected that this reduction will continue for the next 30 to 40 years,” the IUCN Red List said on its website.

“Kibale National Park is probably the last stronghold of chimpanzees in the country of Uganda,” Langergraber said. “We don’t know how exactly how many chimps are there.”

Chimpanzees are hard to count. They live in communities with permanent members, but it’s like a university — you never see the whole community in one place at one time. Chimps make nests every night to sleep in. Counting the nests has been the way the population size is estimated — anywhere from 800 to 1,000 in Kibale.

“We know these nest surveys aren’t terribly accurate,” Langergraber said. “One of the things I’m doing in my research is to really find out how many chimpanzees are in Kibale National Park. We’re doing that through a genetic census.”

Park anti-poaching patrols pick up chimp feces on their patrols. Langergraber does genetic analyses of the samples.

“Then you get a genetic fingerprint for that individual,” he said. “We’re doing this over time. It’s a long-term process. We’ve been doing it for a couple of years now. Based on how many times you’re collecting the same individual over again, versus an entirely new individual you haven’t seen before, you can get an estimate of the population size.”

It’s much more accurate than counting nests.

“Our preliminary results suggests there may be twice as many chimpanzees living in Kibale National Park as we previously thought,” he said. “They’re doing well in Kibale, for now.”

Poaching is widespread throughout the whole park. Eating chimpanzee is actually taboo in this part of Uganda, but poachers go after bush pig and duikers — tiny forest antelope — and chimps become caught in the snares. “They’ll spear it and feed it to their hunting dogs,” Langergraber said. “Poaching has increased, especially for larger game, like elephants.”

The research presence in Ngogo wards poachers away from that area of the park. Two three-person snare-removal teams — ex-poachers lured from the life with good stable jobs — patrol Ngogo in conjunction with local wildlife-authority officers.

A scientist in a forest. “Our guys work with the (Uganda Wildlife Authority) rangers, who have guns, which really helps to protect the park,” said Langergraber (pictured left). The project hopes to hire a third team for patrols.

Research with chimpanzees is long-term. Chimps are long-lived and slow-producing — one infant every five years.

“That makes them very difficult to study,” Langergraber said. “Just now chimpanzee researchers have been out there long enough to really document the whole lifespans of chimpanzees. In particular one of the things we’re interested in in evolutionary biology is what determines which individuals are reproductively successful. ... Things like that are important stuff that’s coming up now and in the future.”

There are multiple chimps at Ngogo more than 60 years old, including one very rare great-grandmother.

“We’ve got a bunch of old ladies running around the forest there,” Langergraber said. “It’s a good place to be a chimp, Ngogo.”