Dan Luo’s new DNA structures could unleash the power of polymers in the fight against bacterial infection, cancer, and even AIDS.

By Glenn Scherer

IN a stunning scientific advance, James Watson and Francis Crick in 1953 revealed the hidden structure of DNA. They proved that nature’s elegant and ingenious double helix is programmable and is the ultimate molecular building block of life.

Now Dan Luo, an assistant professor in Cornell’s Department of Biological and Environmental Engineering (BEE), is doing pathfinding work to extend DNA’s usefulness. He is looking beyond DNA’s purpose as genetic building block, showing it to be a versatile generic building block—an engineering material with near limitless potential.

Luo’s work in nucleic acid engineering—a term he coined just three years ago—could help revolutionize biological engineering at the nanotechnology level. In the short term, the sturdy twisted rope of the DNA molecule could be synthesized to build super-small nanomaterials and nanodevices with amazing biomedical applications, including precise multi-drug delivery systems to treat AIDS or cancer. Further in the future, such materials could be shaped into unimaginably powerful nanomachines and nanocomputers.

“Great potential exists beyond DNA’s use as a genetic material, in using it as a generic engineering material,” asserts Luo, “That’s because we now have the tools at the molecular level, such as enzymes, to design DNA molecules in a true engineering sense, with exacting control over their shape and architecture.”

Luo sees DNA as one of nature’s ultimate polymers, a large molecule comprising many smaller ones linked together, synthesized-to-order through the controlled repetition of those smaller structures. In the modern macro-world, long-chain polymers, such as plastics, revolutionized 20th century society, allowing for the fabrication of complex materials with tailor-made properties—everything from nylon to Plexiglas—to fit thousands of industrial and technological applications.

What is required to achieve such a goal on the nano-level, says Luo, is a change in perspective: We must view DNA not as a means of storing genetic information, but rather as a nano-sized LEGO® block for building new molecules. “DNA through its natural evolution has become a material with self-programming ability. In other words, one DNA strand can recognize its complementary strand. The bonding between these two strands is very specific,” he explains. And these same qualities, which make DNA a perfect information storage molecule, also make it a versatile polymer.

There are, however, major hurdles to DNA’s use as nanotechnology building block: First, all DNA molecules in nature are made only in linear or circular forms, which greatly inhibits their design potential. Imagine, for example, how limited the engineering possibilities of LEGO blocks would be if they only came in stick or circle shapes. But if we were able to create other basic configurations, “Y” or “X” shapes for example, in a controlled manner, the engineering potential would expand exponentially.

The other two limiters to DNA’s use as a nano-polymer are low yield and purity. “When Dr. Nedarin Seeman of NYU first tried to use DNA for nanotechnology purposes in the late 1980s, he made some very impressive structures, such as DNA cubes,” relates Luo. “But while that provided a proof of concept, his yield was less than one percent,” making these structures impractical for immediate application. In a sense, Seeman was hand-making a Stradivarius violin. But what is needed for most biomedical and other applications is accurate mass production of new DNA nanoscale structures—like making Model-T cars on an assembly line.

In fact, Luo and his Cornell collaborators may be well on the way to becoming the Henry Fords of nucleic acid engineering. They have taken huge strides to overcome all three of the limiting factors.

The accomplishment gaining the most attention is the making of dendrimer-like DNA molecules by Luo’s team. A dendrimer (from the Greek dendra for tree) is an artificially synthesized molecule built up from many branched units called monomers. Creating novel dendritic architecture out of DNA is especially challenging since the researchers are working at the scale of nanometers—a nanometer is 10-9 meter or a millionth of a millimeter.

“All the molecules in the world can be classified into four different architectures: linear, branched, networked (like mesh), and dendritic,” Luo clarifies. “On the macro-level, dendritic structures are one of the most abundant architectures in nature. You see them in tree shapes, root shapes, leaf veins, and river deltas. But at the molecular nano-scale, there are no known naturally occurring dendritic molecules.”

In January 2004, Luo and his associates publicized their creation of the first-ever dendrimer-like DNA molecule synthesized in a controlled manner with a high yield that is very pure.

The structure is built from a novel, laboratory-designed, Y-shaped DNA molecule, with specific “sticky” ends that enable it to connect to other very specific Y-DNA molecules via their sticky ends in a highly controlled and enzyme-catalyzed fashion. These basic Y-DNA building blocks can be “grown” into varied dendrimer-like structures that look like snowflakes and can be seen by the electron microscope. This achievement greatly expands the nanoscale LEGO building set that had only contained sticks and circles, giving it new design potential.

Since this success, Luo and his colleagues have gone on to synthesize “X” and “T” shaped DNA molecules, without any genetic purpose, but giving their nano-DNA architecture infinite design versatility.

Luo’s team has already begun developing applications for dendrimer-like DNA. “It is very important to note that we can make our dendrimers ‘anisotropic,’ that is, we can make them different in a precisely controlled way,” says Luo. “Most branched polymers are ‘isotropic,’ so the branches must either all be exactly the same, or all different in a random manner. But we can precisely control which branch is which. That means that we can add molecules to the structure in a totally directed manner.” If, for example, Luo wanted to bond three different drugs to a dendrimer-like DNA structure, he could design different-sized sockets or chemical hooks to hang each drug on, a process called conjugation.

Currently Luo is developing a nano barcode using dendrimer-like DNA to recognize specific bio-molecules in the human body or environment—a process that works something like a cash register reading barcodes on products in a supermarket. “With a DNA barcode we could detect thousands of different genes or pathogens in the body simultaneously. The DNA molecule used this way becomes like a gene chip in solution,” says Luo. Another promising application for DNA barcodes is in environmental detection, creating sensors at the nano-level to simultaneously detect many types of microorganisms in soils, a project Luo is working on with BEE colleague Professor Larry Walker.

Many diseases are caused and advanced by several factors. So drugs (including genes) that are aimed at a single target have limited success in treating complicated diseases such as cancers. Conceptually, it would be more effective to treat tumors with multiple drugs, ranging from small drugs to macromolecular drugs such as gene therapy, that are directed against not a single, but numerous targets.

The anisotropic characteristic of dendrimer-like DNA makes it an ideal structure for delivering multiple macromolecular drugs to a cell in precise doses. But it is extremely difficult for macromolecules such as DNA or proteins to enter the cell. There is no way to be certain that several DNA and protein drugs injected into the human body will enter the desired cells in the desired amount. “You must now treat a disease twice, or more, first with gene A, then anti-gene B, then antibody C. But whether drugs A, B, and C all enter the same cell is purely chance,” Luo explains. “It would be revolutionary if we could deliver gene therapy, anti-gene therapy, antibody therapy, and chemotherapy in a controlled manner within one delivery vector.”

But the greatest barrier to a multi macro drug delivery system is the human cell itself. “Cells behave much as a well-guarded building with many security measures to prevent suspicious macromolecules such as DNA from entering.” There are further defenses to prevent alien DNA from getting inside the cell nucleus. “It is harder to deliver DNA from outside of a cell to inside a nucleus than to get a man from Earth to Mars,” jokes Luo.

To develop a working multi-drug delivery vehicle, Luo is studying viruses. “They are the most efficient DNA and protein delivery machines,” he notes. “But of course, they cause disease and are dangerous. So we are dissecting different viruses, removing the components that cause infection, and isolating only those components that are important in delivery to the cell and nucleus. In essence, we’re constructing an artificial virus that is not a virus at all. Our dendrimer-like DNA serves as the architectural core, the scaffold to link those amazing viral delivery proteins onto one vector. So our nanoparticles are artificial and non-infectious, but powerful viral delivery protein assemblies. We call our delivery system Viral and Non-Viral Assembly, the VNA system.”

Such a multi macro drug delivery system could one day be used to accurately convey drug combination cocktails to AIDS patients or to provide, at once, several chemotherapy drugs with antibodies to cancer patients in precise dosages inside the cell.

A designer-DNA multiple delivery system would also be ideal in gene and anti-gene combined therapy, where it is necessary not only to provide damaged cells with copies of good genes, but also to destroy defective genes at the same time.

Such a multiple delivery system could work for bacterial infection too. If a bacteria has developed resistance, an increasingly common problem in today’s hospitals, a DNA-vector could allow treatment of an infection with two or more antibiotics at once.

“We already have promising preliminary results in one DNA multi-drug delivery project,” Luo reveals. “We’ve succeeded in creating a viral-protein-DNA hybrid and are seeing if it can enter a cell or not. It looks so far as if it can. That is a very striking phenomenon.”

“We are also working on a nano-heater,” Luo adds. “It is a means of generating [very localized but intense] heat using dendrimer-like DNA. We think it could be used to inactivate a virus in the body and to deactivate infectious microbes.”

Luo has been intrigued by the mysteries of DNA since his undergraduate days. He obtained his bachelor’s degree from the University of Science and Technology of China in 1989, doing his undergrad thesis on computer simulations that showed how antibacterial protein drugs could alter their structures to bind to DNA.

Working on his Ph.D. at Ohio State University, Luo studied topisomerases, important proteins that can alter the topology of DNA. Topisomerases are produced by cancerous cells in larger amounts, so they are major targets of cancer chemotherapies. “I was trying to understand the protein-DNA binding action with the presence of chemotherapy drugs,” explains Luo. He also worked on a DNA network that could be used in cancer gene therapy, though such a network is currently too large to get inside of a cell and nucleus. Luo did his postdoctoral training at Cornell’s School of Chemical and Biomolecular Engineering, focusing on synthetic DNA delivery systems. He joined the faculty of the Department of Biological and Environmental Engineering in May 2001.

“At Cornell, I started by studying a DNA delivery system that would protect the DNA inside the hostile environment of the cell. I worked with a controlled release polymer,” which is, Luo explains, very much like a time-release drug. “We encapsulated the DNA into this polymer in a microsphere format, then it was gradually released, in a controlled manner. We found that the DNA was indeed protected before it was released, and it maintained its biological and genetic function.”

Different medical situations require drug delivery systems at different scales. Luo has also worked on the nano-scale, micro-scale, and milli-scale DNA delivery systems that were based on silica nanoparticles, chemical dendrimers, nanofabricated electrodes, and other controlled release polymers. “Most of these systems are based on off-the-shelf and isotropic materials,” Luo explains. “I would like to use anisotropic polymers instead to control the design.”

Luo has more recently focused on dendrimer-DNA anisotropic patterning, an important design innovation. DNA, since it can be easily chopped away or dissolved, can act like a nanoscale mold or form. “You can create a DNA pattern, a honeycomb shape or surface, for example,” says Luo. “You coat the surface with other chemicals, then treat it with enzymes, until all the DNA is digested away. The originally occupied pattern of DNA will remain there as a clean pattern, like a stencil.”

Such patterning techniques would have many engineering applications, including the making of nanowires, nanoscale sieves, even nanomachines or nanocomputers that would use DNA “Y,” “X,” or “T” shapes in various controlled combinations to make circuits.

It is difficult, probably impossible, says Luo, to imagine the full scope of DNA nano-material engineering applications today, just as it would have been impossible in 1900 to guess at the ways in which plastic polymers would remake the 20th century with the then-unimagined wonders of nylon and acrylic fibers, polyethylene (used for plastic bags and bottles), and polycarbonate and polyacrylamide (the material used in compact discs). Today’s ubiquitous plastics serve as spacecraft and computer components, as heart valves, and of course LEGOs! Tomorrow’s dendrimer-like DNA applications may stretch across an equal number of mind-boggling horizons. “What is demanded is a broad interdisciplinary approach to achieve the full potential,” says Luo. “We are currently in a quite exploratory stage in biological engineering. In terms of our research and applications, we need to integrate an engineering component, chemistry component, biology and physics components, plus a polymer and drug delivery component.” It’s a very rich and complex cross-pollinating process.

“And of course this is one of the strengths at Cornell. There are almost no walls between departments, and multiple collaborations are easy,” concludes Luo. “I’m very lucky to have so many fine collaborators in other departments, along with a supportive department, and the best undergrad and grad students.” It is through such multi-branched, dendrimer-like linkages that Luo hopes the promise of nucleic acid engineering will be fulfilled.