DNA Nanotechnology Strategy Puts Enzyme Catalysis Within Reach

Using molecules of DNA in an architectural scaffold, scientists have developed a three-dimensional (3D) synthetic enzyme cascade that mimics an important biochemical pathway that could prove important for future biomedical and energy applications. In this latest research, the research team took up the challenge of mimicking enzymes outside the friendly confines of the cell. These enzymes speed up chemical reactions, used, for example, for the digestion of food into sugars and energy during human metabolism.

Arizona State University (ASU; Tempe, USA) scientists, working with colleagues at the University of Michigan (Ann Arbor, USA), have developed the technology for future biomedical and energy applications. The study’s findings were published online May 25, 2014, in the journal Nature Nanotechnology. Led by ASU Prof. Hao Yan, the research team included ASU Biodesign Institute researchers along with colleagues Prof. Nils Walter and postdoctoral fellow Alexander Johnson-Buck at the University of Michigan.

Researchers in the field of DNA nanotechnology, taking advantage of the binding properties of the chemical building blocks of DNA, twist and self-assemble DNA into ever-more imaginative two- and three-dimensional structures for medical, electronic and energy applications.

In the latest cutting-edge approach, the researcher attempted to mimic enzymes outside the friendly boundaries of the cell. These enzymes speed up chemical reactions, used in bodies for the digestion of food into sugars and energy during human metabolism, for example.

“We look to nature for inspiration to build human-made molecular systems that mimic the sophisticated nanoscale machineries developed in living biological systems, and we rationally design molecular nanoscaffolds to achieve biomimicry at the molecular level,” Prof. Yan said, who also directs the Center for Molecular Design and Biomimicry at the Biodesign Institute.

With enzymes, all moving parts must be tightly controlled and coordinated; otherwise the reaction will not work. The moving parts, which include molecules such as substrates and cofactors, all fit into a complex enzyme pocket just like a baseball into a glove. Once all the chemical parts have found their place in the pocket, the energetics that control the reaction become favorable, and swiftly make chemistry happen. Each enzyme releases its product, similar to a baton handed off in a relay race, to another enzyme to carry out the next phase in a biochemical pathway in the human body.

For the new study, the researchers chose a pair of universal enzymes, glucose-6 phosphate dehydrogenase (G6pDH) and malate dehydrogenase (MDH), that are important for biosynthesis—making fats, amino acids, and nucleic acids vital for all life. For instance, flaws found in the pathway are known to cause anemia in humans. “Dehydrogenase enzymes are particularly important since they supply most of the energy of a cell,” said Prof. Walter. “Work with these enzymes could lead to future applications in green energy production such as fuel cells using biomaterials for fuel.”

In the pathway, G6pDH uses the glucose sugar substrate and a cofactor called NAD to peel off hydrogen atoms from glucose and transfer to the next enzyme, MDH, to continue and produce malic acid and generate NADH in the process, which is used for as a key cofactor for biosynthesis.

Re-creating this enzyme pair in the test tube and having it work outside the cell is a big hurdle for DNA nanotechnology. To meet the challenge, they first constructed a DNA scaffold that looks like several paper towel rolls glued together. Using a computer program, they were able to engineer the chemical building blocks of the DNA sequence so that the scaffold would self-assemble. Next, the two enzymes were attached to the ends of the DNA tubes. In the middle of the DNA scaffold, they attached a single strand of DNA, with the NAD+ tethered to the end like a ball and string. Dr. Yan refers to this as a swinging arm, which is long, and flexible enough to rock back and forth between the enzymes.

Once the system was developed in vitro by heating up and cooling the DNA, which leads to self-assembly, the enzyme parts were added in. They confirmed the structure using a high-powered an atomic force microscope (AFM), which can see down to the nanoscale.

Similar to architects, the scientists first built a full-scale model so they could test and measure the spatial geometry and structures, including in their setup a tiny fluorescent dye attached to the swinging arm. If the reaction takes place, they can measure a red beacon signal that the dye gives off—but in this case, unlike a traffic signal, a red light means the reaction works. Next, they tried the enzyme system and found that it worked just the same as a cellular enzyme cascade. They also measured the effect when varying the distance between the swinging arm and the enzymes. They discovered that there was a perfect spot, at 7 nm, where the arm angle was parallel to the enzyme pair.

With a single swinging arm in the in vitro system working just like the cellular enzymes, the researchers added arms, assessing the limits of the system with up to four added arms. They were able to show that as each arm was added, the G6pDH could keep up to make even more product, while the MDH’s limit was two swinging arms. “Lining enzymes up along a designed assembly line like Henry Ford did for auto parts is particularly satisfying for someone living near the motor city Detroit [MI, USA],” said Prof. Walter.

The research also creates new strategies where biochemical pathways can be replicated outside the cell to develop biomedical applications such as detection methods for diagnostic platforms. “An even loftier and more valuable goal is to engineer highly programmed cascading enzyme pathways on DNA nanostructure platforms with control of input and output sequences. Achieving this goal would not only allow researchers to mimic the elegant enzyme cascades found in nature and attempt to understand their underlying mechanisms of action, but would facilitate the construction of artificial cascades that do not exist in nature,” said Prof. Yan.

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