Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Chemical cages: New technique advances synthetic biology

10.02.2016

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.


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.

Credit: Jason Drees: The Biodesign Institute at Arizona State University

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," Yan says. "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 as well as the Department of Chemistry, Rutgers and the Department of Chemistry, Single Molecule Analysis Group, 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 accompanying animation). 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.

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.

###

Nano-caged Enzymes with Enhanced Catalytic Activity and Increased Stability against Protease Digestion

Zhao Zhao1,3, Jinglin Fu4, Soma Dhakal5, Alexander Johnson-Buck5, Minghui Liu1, Ting Zhang4, Neal W. Woodbury2,3, Yan Liu1,3, Nils G. Walter5 and Hao Yan1,3

1Center for Molecular Design and Biomimetics, 2Center for Innovations in Medicine, the Biodesign Institute, 3School of Molecular Sciences, Arizona State University, Tempe, AZ 85287. 4Department of Chemistry, Rutgers University-Camden, Camden, NJ, 08102. 5Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, MI, 48109, USA.

Richard Harth | EurekAlert!

Further reports about: Biomimetics DNA enzyme nanocages synthetic synthetic biology

More articles from Life Sciences:

nachricht 'Y' a protein unicorn might matter in glaucoma
23.10.2017 | Georgia Institute of Technology

nachricht Microfluidics probe 'cholesterol' of the oil industry
23.10.2017 | Rice University

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Salmonella as a tumour medication

HZI researchers developed a bacterial strain that can be used in cancer therapy

Salmonellae are dangerous pathogens that enter the body via contaminated food and can cause severe infections. But these bacteria are also known to target...

Im Focus: Neutron star merger directly observed for the first time

University of Maryland researchers contribute to historic detection of gravitational waves and light created by event

On August 17, 2017, at 12:41:04 UTC, scientists made the first direct observation of a merger between two neutron stars--the dense, collapsed cores that remain...

Im Focus: Breaking: the first light from two neutron stars merging

Seven new papers describe the first-ever detection of light from a gravitational wave source. The event, caused by two neutron stars colliding and merging together, was dubbed GW170817 because it sent ripples through space-time that reached Earth on 2017 August 17. Around the world, hundreds of excited astronomers mobilized quickly and were able to observe the event using numerous telescopes, providing a wealth of new data.

Previous detections of gravitational waves have all involved the merger of two black holes, a feat that won the 2017 Nobel Prize in Physics earlier this month....

Im Focus: Smart sensors for efficient processes

Material defects in end products can quickly result in failures in many areas of industry, and have a massive impact on the safe use of their products. This is why, in the field of quality assurance, intelligent, nondestructive sensor systems play a key role. They allow testing components and parts in a rapid and cost-efficient manner without destroying the actual product or changing its surface. Experts from the Fraunhofer IZFP in Saarbrücken will be presenting two exhibits at the Blechexpo in Stuttgart from 7–10 November 2017 that allow fast, reliable, and automated characterization of materials and detection of defects (Hall 5, Booth 5306).

When quality testing uses time-consuming destructive test methods, it can result in enormous costs due to damaging or destroying the products. And given that...

Im Focus: Cold molecules on collision course

Using a new cooling technique MPQ scientists succeed at observing collisions in a dense beam of cold and slow dipolar molecules.

How do chemical reactions proceed at extremely low temperatures? The answer requires the investigation of molecular samples that are cold, dense, and slow at...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

3rd Symposium on Driving Simulation

23.10.2017 | Event News

ASEAN Member States discuss the future role of renewable energy

17.10.2017 | Event News

World Health Summit 2017: International experts set the course for the future of Global Health

10.10.2017 | Event News

 
Latest News

Microfluidics probe 'cholesterol' of the oil industry

23.10.2017 | Life Sciences

Gamma rays will reach beyond the limits of light

23.10.2017 | Physics and Astronomy

The end of pneumonia? New vaccine offers hope

23.10.2017 | Health and Medicine

VideoLinks
B2B-VideoLinks
More VideoLinks >>>