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March 21, 2013

DNA to Hang Your Hat On

Scientists are coming up with amazing new uses for DNA origami.

DNA to Hang Your Hat On

To progress nanotechnology, scientists will continue to exploit DNA’s physical and chemical properties as well as its self-assembly in inventive ways. [© Jasenka - Fotolia.com]

  • Origami DNA is DNA folded to create two and three dimensional shapes at nanoscale. In laboratories using the technology, originally developed by Paul Rothemund, Ph.D., at Cal Tech, it could eventually prove useful for developing novel molecular sensors for diagnostic devices, molecular drug containers, and/or delivery systems, and to order entire enzymatic reactions. Laboratories are using DNA scaffolding, or more elaborately structured DNA, for a variety of new applications.

    Dr. Rothemund developed his simplified “one-pot” method for using numerous short single strands of DNA to direct the folding of a long, single strand of DNA, viral DNA in the original incarnation of the method, into desired shapes in 2006. His relatively simple process, he explained, allowed complexity formerly achievable only by “bottom-up” fabrication, an approach involving rounds of synthesis and purification and requiring days to weeks.

    With the method, he produced shapes roughly 100 nm in diameter with a spatial resolution of about 6 nm, assembling six different shapes, such as squares, triangles, and five-pointed stars. “I show that the method not only provides access to structures that approximate the outline of any desired shape, but also enables the creation of structures with arbitrarily shaped holes or surface patterns composed of more than 200 individual pixels,” he said, dubbing the method “scaffolded DNA origami”.

    Dr. Rothemund envisioned that an “obvious” application of patterned DNA origami would be the creation of a “nanobreadboard” to which diverse components could be added, such as the attachment of proteins to allow novel biological experiments to model complex protein assemblies.

  • 3D DNA Origami

    Scientists have since created multiple variations on Dr. Rothemund’s theme. Last November, investigators led by Wyss Institute faculty member Bryan Wei, Ph.D., reported that they created scaffold-free 3D structures starting with smaller DNA bricks, about 32 bases long, which changes the orientation of every matched-up pair of bricks to a 90 degree angle, giving every two bricks a 3D shape.

    Wei’s team focused on using short strands of synthetic DNA (SSTs), avoiding the use of a long scaffolding strand. Each SST is a single short strand that interlocks with another tile if it has a complementary sequence of DNA. If there are no complementary matches, the blocks do not connect. Thus, a specific collection of tiles can assemble itself into predetermined shapes through a series of interlocking local connections.

    Dr. Wei told GEN, “The scaffold-free approach liberates us from the long single-strand DNA obtained from viruses. Therefore, the components of our approach are 100% synthetic. Because we are using synthetic strands for the self-assembly, we are not limited by natural nucleic acids. For example, we adopted L-DNA, the mirror structure of natural D-DNA, for self-assembly. In that case, the resulting structure was resistant to DNA degradation and supposed to have limited immunogenicity. These properties are appealing for DNA structures serving as drug delivery vectors.”

  • Drug Delivery

    The research group of Björn Högberg, Ph.D., at the Karolinska Institutet has focused on techniques to bring DNA devices to practical use in medicine.

    The Karolinska team uses a long single-stranded DNA molecule or scaffold, and then mixes the scaffold with about 200 short, single-stranded oligonucleotides, or staples. As all of the short-staple oligos hybridize to the long single-stranded scaffold during a slow thermal annealing, nanoscale shapes of all kinds can be produced. After self-assembly of the DNA nanostructures, the exact position of each oligo is known.

    In 2012 the scientists reported the successful design and testing of two DNA origami nanostructures to deliver the anthracycline drug doxorubicin (Dox) to three different human breast cancer cell lines. Because anthracyclines' mechanism of action in cancer therapy is to intercalate DNA, and since DNA nanotechnology allows for such a high degree of customization, the scientists hypothesized that these properties would allow “tuning” of the DNA nanostructures for optimal delivery of the drug to the cells.

    By tuning the nanostructure design, they reported, they could also tune the encapsulation efficiency and the release rate of the drug, as well as increase the cytotoxicity and lower the intracellular elimination rate when compared to free drug.

    The scientists concluded that “the promising release kinetics and cytotoxicity of the Dox/T-Nano system, in combination with the well-known flexibility of the DNA origami method to decorate the structures with targeting ligands make the DNA structures a promising potential candidate platform for active targeting of anticancer nanostructures.”

  • Enzyme Cascades

    University of California Riverside scientists have used DNA nanostructures to create spatially organized enhanced enzymatic cascades. Ian Wheeldon, Ph.D., assistant professor in the department of chemical and environmental engineering, and his group develop bioanalytical devices for applications including medical diagnostics and environmental monitoring. These devices, for example, could take the form of enzymatic pathways organized on DNA scaffolding for bioassays.

    In a recent paper in American Chemical Society Catalysis, Dr. Wheeldon’s team noted DNA’s properties can be used to create precisely defined multidimensional shapes with molecular-level control over structural and chemical features, potentially allowing creation of multienzyme cascades with well-defined spatial organization.

    The team investigated the interactions between enzyme substrates and DNA scaffolds using a model system of horseradish peroxidase (HRP) assembled on a nanoscale DNA triangle.

    “We used [HRP] as a model system to look at a series of substrates because this enzyme oxidizes a wide range of chemically distinct substrates that are commonly used in analytical assays, and all varying in their interactions with DNA,” Dr. Wheeldon said.

    He continued, “We were trying to answer the question, does DNA as a scaffold make a difference to the system? A group of us is looking at using DNA as a way to organize enzymes into a pathway, for example, an efficient pathway of three enzymatic reactions. DNA is one of the only systems where we can get down to tens of nanometers and that we can use to organize pathways.

    “In determining how tightly the substrates bind to DNA, we found that those that bound tightly and those that bound weakly had no effect on catalysis, but when they bound at intermediate strength, it increased catalysis. We think this occurs because as these substrates repeatedly bind to and release from the DNA scaffold, the substrate is kept at high levels around the enzymes, thereby driving high rates of catalysis.

    “We are really more enzyme than DNA people, but if we are going to use DNA as a scaffold, we need to understand what it means in terms of the rest of the system.”

    With spatially controlled positioning of functional materials by self-assembly remains a key goal of nanotechnology, scientists will continue to exploit DNA’s physical and chemical properties, as well as its self-assembly in inventive ways.

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