Using an approach called Origami of DNA, Caltech scientists have developed a technique that could lead to cheaper reusable biomarker sensors to quickly detect protein proteins in body fluids, eliminating the need to send samples to laboratory centers for tests.
“Our work provides a proof of concept that shows a route to a single step method that could be used to identify and measure nucleic acids and proteins,” says Paul Rothemund (BS ’94), an associate visitor in Caltech in computing and Computing and mathematical sciences, and calculation and neuronal systems.
An article that describes the work recently appeared in the magazine Proceedings of the National Academy of Sciences. The main authors of the newspaper are the former postdoctoral scholar of Caltech, Byoung-Jin Jeon, and the current graduate student Matteo M. Guareschi, who completed the work in the Rothemund laboratory.
In 2006, Rothemund published the first article about Origami de ADN, a technique that provides simple but exquisite control over the design of molecular structures in the nanoscale that uses nothing more than DNA.
Essentially, DNA origami allows long DNA threads to be folded, through self -assembly, in any desired form. (In the 2006 article, Rothemund used the technique to create smiling miniature DNA faces that measure 100 nanometers wide and 2 thick nanometers). Researchers begin with a long DNA chain, scaffolding, in solution. Because the nucleotide bases that form the DNA are bind to the scaffold at any end in known locations. Those short and aggregate pieces of DNA bend the scaffold and shape it, acting as “staples” that keep the structure together. The technique can be used to create forms that range from a map of North and South America to transistors to Nanoscala.
In the new work, Rothemund and his colleagues used DNA origami to create a structure similar to a lilypad: a flat circular surface of approximately 100 nanometers of diameter, tied by a DNA linker to a gold electrode. Both Lilypad and the electrode have short DNA strands available to join an analyte, a molecule of interest in the solution, be it a DNA molecule, a protein or an antibody. When the analyte joins those short strands, the Lilypad is lowered to the gold surface, carrying 70 reporters in the Lilypad (indicating that the directed molecule is present) in contact with the gold surface. These reporters are redox reactive molecules, which means that they can easily lose electrons during a reaction. Then, when they approach enough to an electrode, an electric current can be observed. A stronger current indicates that there is more of the molecule of present interest.
Previously, a similar approach was developed to make biosensors using a single DNA chain instead of a DNA origami structure. That previous work was directed by Kevin W. Plaxco (PHD ’94) of UC Santa Barbara, who is also the author of the current document.
Caltech Guareschi points out that Lilypad’s new origami is large compared to a single DNA chain. “That means that it can adjust to 70 reporters in a single molecule and keep them away from the surface before the union. Then, when the analyte is united and the Lilypad reaches the electrode, there is a large signal gain, which makes the change is easy to detect. ” Guareschi says.
The relatively large size of the Lilypad origami also means that the system can easily accommodate and detect larger molecules, such as large proteins. In the new article, the team showed that the two short DNA threads in the Lilypad and the gold surface could be used as adapters, which makes it a protein sensor instead of DNA. At work, the researchers added the biotin vitamin to those short DNA threads to convert the system into a sensor for the streptavidine protein. Then they added a DNA sister, a DNA chain that can join a specific protein; In this case, they used a suitcase that binds to a protein called growth factor derived from BB platelets (PDGF-BB), which could be used to help diagnose diseases such as cirrhosis and intestinal inflammatory disease.
“We simply add these simple molecules to the system, and you are ready to feel something different,” says Guareschi. “It is large enough to accommodate whatever I throw, that it could be aptmers, nagods, antibody fragments, and it does not need to be completely redesigned every time.”
Researchers also show that the sensor can be reused several times, with new adapters added each round for different detections. Although the performance is degraded slightly over time, the current system could be reused at least four times.
In the future, the team expects the system to also be useful for proteomics, studies that determine which proteins are in a sample and what concentrations. “I could have multiple sensors at the same time with different analytes, and then I could wash, change the analytes and solve it. And I could do it several times,” says Guareschi. “In a few hours, you can measure hundreds of proteins using a single system.”
The additional authors of the article, “electrochemical detection of DNA and proteins based on modular DNA” are Jaimie M. Stewart of UCLA; Emily Wu and Ashwin Gopinath of MIT, Netzahualcóyotl Arroyo-Curás of the Faculty of Medicine of the Johns Hopkins University, Philippe Dauphin-Ducharme of the Sherbrooke University in Canada; and Philip S. Lukeman of St. John University in New York.
The team used manufacturing equipment at Kavli Nanoscience Institute in Caltech. The work was supported by the Army Research Office, the Naval Research Office, the National Foundation of Sciences and the Life Science Research Foundation supported by Merck Research Laboratories.