Using DNA molecules acting as an architectural scaffold, scientists were able to construct a XNUMXD artificial enzymatic array that mimics an essential biochemical pathway that could be important for future applications in the fields of biomedicine and energy.
![Schematic description of the system with two enzymes integrated in a common biochemical pathway of proton transfer. [Courtesy: Biodesign Institute at ASU]](https://www.hayadan.org.il/images/content3/2014/07/73633.jpg)
As part of a long-awaited breakthrough, a research team faced the challenge of imitating the activity of enzymes outside of their normal friendly environment - inside the cells themselves. These enzymes accelerate chemical reactions that occur in our body and are used to break down food into sugars and convert them into energy during metabolism. Arizona State University scientists have developed a XNUMXD artificial enzymatic array that mimics an essential biochemical pathway that could be important for future applications in biomedicine and energy. The findings were published in the scientific journal Nature Nanotechnology.
Research in the field of DNA nanotechnology takes advantage of the binding properties of the chemical building blocks of DNA molecules, while twisting and self-assembling these molecules to build creative two- and three-dimensional structures for medical, electronics and energy applications.
"We turned to nature to receive inspiration from it for the construction of man-made molecular systems that could imitate the sophisticated molecular nanomachines that have developed in biological systems, and we designed molecular nanoscaffolds in an orderly manner to achieve the replication of biology at the molecular level," said the lead researcher.
In the realm of enzymes, all the moving parts must be highly coordinated and controlled, otherwise the reaction will not occur at all. The moving parts, which include molecules such as reactants and cofactors, all fit with great precision into a complex enzyme niche. Once all the chemical parts find their place in the niche, the energies controlling the reaction become favored, causing the chemical reaction to occur faster. Each enzyme releases its product to another enzyme which in turn is responsible for the next step in the biochemical pathway in the human body, similar to a baton being passed from sprinter to sprinter in a relay race.
In the current study, the researchers chose a pair of universal enzymes, glucose-6 phosphate dehydrogenase (G6pDH) and malate dehydrogenase (MDH), which are important for the biosynthesis of the amino acids, lipids and nucleic acids that are all essential to life. For example, defects occurring in this pathway cause anemia in humans. "Enzymes from the dehydrogenase group are particularly important since they provide most of the cell's energy," explains the lead researcher. "Research in the field of these enzymes could lead to future developments of applications for the production of green energy, for example, the development of fuel cells that use bio-materials to produce fuel."
As part of this biochemical pathway, G6pDH uses the substrate of the sugar glucose and with the help of the co-factor NAD removes hydrogen atoms from the glucose and then passes them to the next enzyme, MDH, which in turn creates fruit acid and the co-factor NADH, which itself is an essential component in biosynthesis processes. Preparing a pair of these enzymes in vitro and obtaining the possibility of their required activity are a serious challenge in the field of DNA nanotechnology.
In order to meet this challenge, the researchers first prepared a DNA scaffold that looks like a number of pages rolled together into a cylinder. With the help of a computer program, the researchers were able to select the most appropriate chemical building blocks of the DNA sequence so that the scaffold would be built by self-assembly. In the next step, the two separate enzymes were bound to the ends of the DNA coil. In the center of the DNA scaffold, the researchers added a single strand of DNA to the end of which the co-factor NAD was attached, like a ball tied to a rope. The researchers define this part as a rocking arm, which is long, flexible and dedicated enough to jump between the two separate enzymes.
After the system was built in a test tube by heating and cooling the DNA, which leads to its self-assembly, the enzyme components were added to the test tube. The researchers verified the overall structure with a sophisticated microscope (AFM) capable of distinguishing nanostructures. Like architects, the scientists first built a full-scale model so they could examine and measure the spatial geometry and various structures, adding to the array a tiny fluorescent material attached to the rocking arm. If the reaction does occur as expected, the researchers are able to measure the signal of red radiation emitted by the fluorescent substance. Next, they discovered that the enzyme system works just like the cellular enzyme system. The researchers also measured the effect of changing the distance between the swinging arm and the enzymes - they found that the optimal distance is 7 nanometers, at the point where the angle of the arm is parallel to the pair of enzymes.
After the researchers discovered that the test tube system with one arm works exactly like the biological system, they decided to add arms in order to test the limitations of the system with up to 4 arms. They showed that each additional arm allows increased activity of the G6pDH receptors, while the second enzyme (MDH) reached its maximum activity when there were only 2 arms in the system.
The new method paves the way for a bright future where it will be possible to replicate biochemical pathways outside cells in order to develop biomedical applications such as detection methods for diagnosing diseases and medical conditions. "A loftier and more useful goal would be the construction of pre-programmed enzyme pathways on top of DNA nanostructures while controlling the input materials and the output products. Achieving this goal will not only allow researchers to imitate the elegant enzymatic pathways found in nature while understanding the underlying mechanisms, but they will also be able to advance the construction of artificial arrays that are not found in nature at all," said the lead researcher.
One response
m”k the chemical building blocks