SEOUL, South Korea, Sept. 28, 2017 /PRNewswire/ -- Cells recognize external physical and chemical signals, and maintain their homeostasis by responding to these signals via reversibly* controlling the activity of signaling proteins. For example, the cell begins to divide when proteins involved in a cell cycle are activated, and after the division, the activity of these proteins are suppressed. If this reversible reaction is not properly regulated, the cell can be constantly being divided, causing diseases like cancer.
*Reversible reaction: a reaction that can proceed in either forward or inverse direction according to the reaction condition in a chemical reaction.
As such, most of the cellular process by enzymes occurring in the cell can be regulated as desired if the enzyme activity can be artificially and reversibly regulated. The most common method to manipulate the enzyme activity involves inserting the effector domain that can respond to the external physical and chemical stimuli (pH, temperature, light, etc.), but it can be used only when the structure and function of the relevant enzyme is properly understood.
The team led by Dr. So Yeon Kim of Center for Theragnosis in Biomedical Research Institute at Korea Institute of Science and Technology (KIST, President Byung Gwon Lee) has developed a DNA nanoswitch that can be used universally regardless of the structure and function of the enzyme that needs to be controlled. The team announced that they used a DNA nanostructure as a cage to encapsulate the enzyme of interest and have successfully controlled the enzyme activity artificially, reversibly and repeatedly.
The team at KIST designed the DNA sequence so that the desired enzyme is located inside of the tetrahedron-shaped DNA nanocage and inserted pH-sensitive sequence into one side of the tetrahedron. By doing so, they can control the enzyme accessibility to the surrounding environment via pH-dependent opening and closing of the DNA tetrahedron nanocage.
In the conventional method, the cage-like nanostructure was mostly used to encapsulate the enzyme, and the enzyme was physically released by an extra stimulus. Therefore, it was difficult to induce the reverse reaction by recapturing released protein in solution. In other words, the enzyme activity cannot be repeatedly regulated in both direction. However, the newly developed DNA nanoswitch allows to reversibly control the activity because the enzyme of interest is covalently attached inside of a DNA nanocage.
The research team found that when the enzyme (RNase A**)is confined in the newly developed DNA nanocage, both the accessibility to other proteins in the solution and the activity of RNaseA were all suppressed, whereas when the nanocage was opened by the pH change, both the accessibility and activity of RNase A increased. By measuring the enzyme activity while continuously changing the pH, they discovered that the activity can be repeatedly controlled.
**RNAse A : Enzyme that degrades RNA into smaller components. RNAse A is quite stable enzyme, and its activity is maintained in a broad range of temperature (4-70 degrees Celsius) and pH (4.0-9.0). It can be used to kill cancer cells by decomposing RNA in cancer cells.
"The DNA nanoswitch developed through this research can be applied to reveal the unknown biophysical properties of proteins, when operated at a single-molecule level." Dr. So Yeon Kim of KIST said, "It can be used as a enzyme transporter within a cell. Which means the movement of the cell, cell cycle, and even the fate of the stem cell can be controlled precisely, depending on the enzyme of interest. I hope that it will help to develop new concept for cell therapy."
This research was supported by the KIST institutional grant and the National Research Foundation of Korea funded by the Korean Ministry of Science, ICT, and Future Planning. The results were published online via 'ACS Nano' (IF = 13.942), an international academic journal published by ACS, on 28, August 2017.
The enzyme of interest is covalently attached inside of the DNA nanocage. The DNA nanocage is designed to open and close according to pH change by inserting pH-sensitive sequence into one side of the DNA nanocage tetrahedron, and therefore the accessibility and activity of enzyme can be reversibly regulated by pH change.
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SOURCE KIST (Korea Institute of Science and Technology)