DNA Origami proposes a route to reusable, multifunctional biosensors

Using an approach called DNA origami, Caltech scientists have developed a technology that could lead to cheaper and reusable biomarker sensors for rapid detection of proteins in body fluids, and for testing purposes Eliminate the need to send samples to the lab center.

“Our research provides proof of concepts that show the way to a single step method that can be used to identify and measure nucleic acids and proteins,” says visiting associate for Computing and COLPING AND ANCITECH. says Paul Rothemund (BS ’94). Mathematical Science, and Computation and Neural Systems.

A paper describing this work was recently published in the Proceedings magazine of the National Academy of Sciences. The lead authors of this paper are former Caltech Postdoctoral Dr. Biung Jinjung and current graduate student Matteo M. Guareschi, who completed work in the lab of Rosemundo.

In 2006, Rothemund published his first paper on DNA Origami. This is a technique that provides simple yet exquisite control over the design of molecular structures at the nanoscale, which is nothing but DNA.

Essentially, DNA origami allows long strands of DNA to fold into any shape through self-assembly. (In a 2006 paper, Rothemund used this technique to create miniature DNA smiley faces 100 nanometers wide and 2 nanometers thick).

Researchers start with long DNA in solution, the scaffolding strand. Because the nucleotide bases that make up DNA are bound in known ways (adenine binds to thymine and guanine binds to cytosine), scientists know that they bind to scaffolds at both ends at known locations. Hundreds of short complementary DNA can be added.

These short DNA fragments act as “staples” that fold the scaffold, give it shape, and hold the structure together. This technique can then be used to create shapes ranging from maps of North and South America to nanoscale transistors.

In the new work, Rothemund and his colleagues used DNA origami to create a flat, circular surface with a diameter of approximately 100 nanometers, connected by a DNA linker to the gold electrode. Both the LilyPad and the electrodes have short DNA strands available to bind to the analyte, whether they are molecules of interest for solution, DNA, protein, or antibody molecules that are available for binding to the analyte.

When the analytes bind to their short chains, the lily pad is pulled down to the gold surface, bringing 70 reporter molecules (indicating the presence of the target molecule) into contact with the gold surface. These reporters are redox-reactive molecules, which can easily lose electrons during the reaction. Therefore, if you get close enough to the electrode, a current is observed. A stronger current indicates that many of the molecules of interest are present.

Previously, similar approaches have been developed to create biosensors using a single strand of DNA rather than a DNA origami structure. That previous work was led by Kevin W. Prax (D. ’94), of UC Santa Barbara, who is also the author of the current paper.

Caltech’s Guareschi points out that the new Lilypad origami is larger than a single strand of DNA. “So, 70 reporters can be fitted to a single molecule and moved away from the surface before binding. Then, when the analyte binds and the lily pad reaches the electrode, there is a large signal gain and changes. makes it easier to detect,” Guareschi says.

The relatively large size of Lilypad origami means that the system can easily accommodate and detect large molecules such as large proteins. In a new paper, the team showed that two short DNA strands, a lily pad and a gold surface, can be used as adapters, and that they can be made into a protein sensor rather than DNA.

In the study, researchers added vitamin biotin to their short DNA strands, turning the system into a sensor for the protein streptavidin. Next, we added DNA strands, a DNA aptamer that can bind to specific proteins. In this case, they used an aptamer that binds to a protein called platelet-derived growth factor BB (PDGF-BB).

“Just adding these simple molecules to the system, you’re ready to feel something different,” Guareschi says. “It’s big enough to accommodate anything you throw. It doesn’t have to be completely redesigned every time, like aptamers, nanobodies, antibody fragments.”

Researchers also show that the sensors can be reused several times, with new adapters adding each round for different detection. Performance will drop slightly over time, but the current system can be reused at least four times.

In the future, the team hopes that the system may also be useful for studies that determine proteomics, that is, proteins and concentrations contained in the sample. “You can have multiple sensors at the same time using different analytes, then you can wash, switch and delete the analytes, and you can do that several times “We’ll do that,” Guareschi says. “In a few hours, we can measure hundreds of proteins using a single system.”

Additional authors of the paper, “Modular DNA Origami-Based Electrochemical Detection of DNA and Proteins,” is Jaimie M. Stewart of UCLA. Emily Wu and Ashwin Gopinas of Mitt, netzahualcóyotlarroyo-currás Philip Dauphin Ducalm of Johns Hopkins University School of Medicine and Shelbrook University in Canada. Philip S. Lukeman of St. John’s University in New York;