Engineers Build DNA 'Nanotowers' With Enzyme Tools

"The process works like stacking Legos to make a tower and is animportant step toward creating functional nanostructures out ofbiological materials," said Ashutosh Chilkoti, associate professor ofbiomedical engineering at Duke's Pratt School of Engineering.

The prefix nano means a billionth and refers to the billionth-of-a-meter scale of such structures.

Lastyear, Chilkoti and his team demonstrated an enzyme-driven process to"carve" nanoscale troughs into a field of DNA strands. By combiningthis technique with the new method of adding vertical length to the DNAstrands, they can now create surfaces with three-dimensional topography.

"The development of bio-nanotechnological tools and fabricationstrategies, as demonstrated here, will ultimately allow the automatedstudy of biology at the molecular scale and will drive our discoveryand understanding of the basic molecular machinery that defines life,"said Stefan Zauscher, assistant professor of mechanical engineering andmaterials science.

This research was published online on Sept. 27, 2005, and willbe published in the print Journal of the American Chemical Society(JACS). The article is available at: http://pubs3.acs.org/acs/journals/doilookup?in_doi=10.1021/ja052491z. It is funded by the National Science Foundation.

The authors include Chilkoti, Zauscher, postdoctoral fellow Dominic Chow and graduate student Woo-Kyung Lee.

"Compared with semi-conductor fabrication, bio-nanomanufacturing is inthe stone age. There are few tools for working with bio building blocksthat work well in water, the natural milieu of biomolecules," Chilkotisaid. "And it makes little sense to blindly copy the semi-conductorindustry because their techniques don't work with water-basedmaterials," he said. "So Duke is creating the tools that will makebio-manufacturing possible at an industrial scale."

The team starts with a forest of short DNA strands that cover nanoscalepatches of gold, lithographed onto a silicon substrate. The researchersthen submerge the substrate in a solution that contains the TdTase(terminal deoxynucleotidyl transferase) enzyme, a cobalt catalyst andthe molecular building blocks, called nucleotides, of DNA chains.

Over an hour, the TdTase enzyme grabs the free-floating nucleotides andbuilds nanoscale "towers" above the surface by extending each DNAstrand, increasing its height a hundredfold. In addition, the processworks at room temperature in an incubator that maintains humidity,Chilkoti said.

"Working with water-based biological materials requires ahumidity-controlled environment, but it is a plus for industry thatthis surface-initiated polymerization works at room temperature. Nospecial heating or cooling is needed," he said.

"The process is like a surface-initiated polymerization reaction inpolymer chemistry, with the important difference that it usesbiological materials and is enzymatically catalyzed," adds Zauscher."Developing the tools to harness biological reactions on the molecularscale opens a whole new arena for materials syntheses."

Biologists have known about the TdTase enzyme for decades, but it hasonly been used for a few specialized tasks in molecular biology,Chilkoti said. His group was interested in the enzyme because itdoesn't just copy DNA, it builds DNA.

"Biologists call the TdTase enzyme promiscuous because it just buildsand builds using whatever is available. We now recognize the enzymeoffers us fabulous flexibility for bioengineering. We can use it withany sequence of DNA we need," Chilkoti said.

The Duke team sees enzymes as a rich source of tools forbio-nanomanufacturing. "Enzymes are the body's production factories, soit makes sense to copy nature's tools and use them in much the sameway. We are trying to bring as many different enzymes as possible tobear on the biomanufacturing problem," Chilkoti said. "The newfabrication strategy allows exquisite control over the structure andcomposition of the DNA nanostructures, a prospect that offersinteresting possibilities for bionanofabrication as it allows specificmolecular adapters to be encoded along the vertical direction of theDNA chains," said Zauscher.

Chilkoti said the next step towards bio-nanofabrication is to create alittle crane to pick up, move and place biological molecules in preciselocations on three-dimensional DNA surfaces.

"When we can place molecules in the right configuration, then we canget them to function. At that point, we can design and createbiological machines that accomplish something," he said.

Zauscher and Chilkoti are part of Duke's Center for BiologicallyInspired Materials and Materials Systems (CBIMMS), an interdisciplinaryresearch group dedicated to engineering solution to problems using the"design answers" found in nature. Two major research thrusts includenanomedicine and bio-nanomanufacturing. For more information aboutCBIMMS, visit: http://www.cbimms.duke.edu/