In June, Chad Mirkin, Ph.D., Director of the International Institute for Nanotechnology and the George B. Rathmann Professor of Chemistry, Professor of Medicine, Professor of Materials Science and Engineering, Professor of Biomedical Engineering, and Professor of Chemical and Biological Engineering at Northwestern University in Evanston, IL (USA), will present the keynote address at the 2013 SLAS Asia Conference and Exhibition in Shanghai, China. Titled Nano-flares for the Analysis of Circulating Cancer Cells, Mirkin's presentation will include details about a unique spherical nucleic acid (SNA)-based technology that recently became available to researchers, enabling them to take measurements within live cells that cannot be accomplished with conventional techniques.
But Nano-flares are only part of the story, according to Mirkin, who also serves as a scientific advisor to the Journal of Laboratory Automation (JALA). "I'll be focusing on the importance of the spherical nucleic acid (SNA) architecture and how, on a sequence-to-sequence basis, this structure is fundamentally different from linear nucleic acids. Those differences enable us to create high sensitivity, high selectivity probes for medical diagnostic purposes—and they allow us to get Nano-flares into cells so that we can make intracellular measurements that we couldn't do otherwise," he says. "We're now investigating the use of SNAs as gene-regulation agents for therapeutic purposes, looking at ways to track disease at much earlier stages so we can treat it at much earlier stages, and at the same time, enable more rapid discovery of potential therapeutic agents."
Mirkin joined the faculty of Northwestern in 1991. A synthetic chemist by training, he became interested in the first microscopy tools that allowed researchers to see and manipulate atoms. He then explored the chemistry of miniaturization, and began to see how that approach could impact such fields as materials science, biology and medicine.
"From there, things kind of snowballed and my group began playing with nanoparticles, thinking not about building SNAs for biological purposes, but rather for building what we called programmable atom equivalents—particles that would have programmable bonding by virtue of the type of DNA that was put on their surface," Mirkin explains. "The idea was to use DNA as a construction material, like a chemical-specific ball of Velcro, so we could figure out which particles would bind to one another and which ones wouldn't based on DNA base pairing. But then we discovered that these particles have fantastic properties that make them extremely interesting and useful in molecular and medical diagnostics, and most recently, in gene regulation."
Mirkin refined the technology over the years, during which time his group has built more than 200 different crystal structures with 23 different particle arrangements, many of which have no naturally occurring counterparts. In a presentation at the 2013 AAAS meeting in Boston, he showed how the novel particles might be used to "forge a new periodic table."
"Everything we do now revolves around SNAs," Mirkin says. "All these inventions started with the discovery that if we synthesized DNA strands [oligonucleotides] that had groups that could be tacked onto the surface of a gold particle, we could arrange that DNA into a new format—a spherical form, imbued with properties that are different from their linear cousins."
One of those unique properties is the ability to naturally enter cells. "Linear nucleic acids don't efficiently enter cells, but SNAs comprised of identical sequences go right into cells. They don't require cationic transfection materials or any additional modifications to drive cellular entry," Mirkin notes. His team has shown that SNAs can enter more than 50 cell lines to date, including primary cells, as well as whole organs and cultured tissues.
Once inside cells, SNAs "trigger almost no immune response, as measured by interferon-beta levels, and they resist nuclease degradation, meaning they are stable inside cells," Mirkin says. This makes them an ideal therapeutic platform for targeting tumors in the body in all types of organs from the skin to the brain.
Mirkin initially used gold as the scaffold for SNAs; however, he stresses that other materials—e.g., silver, platinum, a quantum dot—would also work, depending on the desired chemical and physical properties of the final structure.
Mirkin was the 2012 winner of the American Chemical Society's Award for Creative Invention for his work with SNAs. Some of his group's earliest discoveries in that arena have already transitioned from the university to the clinic via the Verigene System, medical diagnostics technology that has led to Food and Drug Administration (FDA)-cleared tests for blood cultures, respiratory viruses, C. difficile and warfarin metabolism, among others. The system is marketed by Nanosphere, a company Mirkin founded in 2000, and it is used in hospitals around the world.
"There are other ways of doing DNA detection or genetics-based analyses, but the very simple and very sensitive diagnostic tests enabled by the SNA architecture allows one to do detection rapidly and at the point-of-care," Mirkin explains. "Now we can think about decentralizing the medical diagnostics industry and moving a lot of the technologies that are at remote labs closer to the point-of-use—in hospitals, in the emergency room and, ultimately, in the doctor's office."
More recently, Mirkin has focused on developing SNAs "as a major research tool," he says. "The fact that SNAs freely enter human cells means that we have the ability to get very large amounts of nucleic acids into cells. That allowed us to think we could make measurements inside cells—of mRNA levels, for example—that we couldn't do with conventional agents that get chewed up too rapidly. To do so, we'd have to not only have the ability to bind an mRNA, but also the ability to trigger an event that gives us some sort of spectroscopic change. And that was the birth of the Nano-flare."
Conventional methods for detecting mRNA involve the interrogation of lysed or fixed cell populations using amplification techniques to detect signals. "SNA-based Nano-flare technology enables mRNA detection in living cells in a non-toxic way, without altering cell cultures during or after incubation," he adds.
The technology is licensed to AuraSense, a company Mirkin cofounded with his Northwestern colleague, C. Shad Thaxton, M.D., Ph.D., which subsequently partnered with Merck Millipore. About 170 different types of Nano-flares became available through Merck Millipore in the fall of 2012, according to Mirkin, under the trademark SmartFlares™. Part of the benefit to potential users is that the company "has made the technology both simple to understand and very easy to use," Mirkin says.
In a demo on its website, Merck Millipore explains that the Nano-flares "consist of gold nanoparticles conjugated to duplexed oligonucleotide sequences. The capture strand is a short oligonucleotide sequence complementary to your RNA sequence. The reporter strand is complementary to the capture strand and is about half its size. It is conjugated to a fluorophore of your choice, which when bound to the capture strand remains quenched by the gold nanoparticle."
Mirkin elaborates: "The gold is important because it quenches the fluorescence so that it is spectroscopically silent. When the Nano-flare gets taken up by a cell, it goes in and finds an mRNA complement that it's been designed to recognize and binds to it. At that point, the reporter strand is released, causing the fluorophore to fluoresce because it is no longer quenched by the gold nanoparticle. So for every mRNA binding event, a fluorophore is released, which then triggers a spectroscopic signaling event.
"When we couple this capability with flow cytometry, for the first time we can measure the genetic content of live cells in real time. We don't have to chew them up, grind them up, release the nucleic acid and use things like real time PCR to make those measurements. That's fantastic, because it means the researcher can now feed a collection of cells whose identities are unknown a specific type of Nano-flare that targets a gene present only in cancer cells, for example, and the cancer cells in the population will selectively light up and the healthy ones won't. One can differentiate stem cells from non-stem-cell-type structures. One can look at how the up- and down-regulation of genes is affected through the addition of a chemical agent for drug screening purposes."
The DNA strands that decorate the gold nanoparticle's surface are important for two reasons, Mirkin continues. "They trigger endocytosis via their interaction with signaling proteins from the extracellular matrix; this allows the nanoparticles to be distributed throughout the cell. They also act as a genetic-regulation mechanism. When they bind to mRNA, they effectively shut down the ability of the cell to produce the specific protein the mRNA encoded for. And the particles also can be synthesized based on siRNA, another potent nucleic acid-based gene-regulation material. Those capabilities lay the groundwork for the next wave—potential therapeutics."
Mirkin and his group are now moving forward in two main areas. First, they are exploring ways to turn Nano-flares from a research technique into a medical diagnostic tool. "For example, we are asking such questions as ‘can we use the technology to fish out a few circulating tumor cells in a blood sample?' We think we could completely change the way breast cancer diagnostics is done by flagging circulating tumor cells with Nano-flares as opposed to doing crude tests that are not very informative because they don't provide genetic information."
His group is also targeting therapies. "We think that these types of SNA architectures, in general, are going to form the basis for a whole new class of therapeutics that we are rapidly developing for a wide range of diseases." For example, recent work in animal models has revealed that SNAs can be used to silence the glioma oncoprotein Bcl2L12 (BC12-like 12) and promote apoptosis in vivo, thereby slowing tumor growth and improving animal survival. In addition, Mirkin's lab has shown that SNAs penetrate all types of tissues, including brain, heart, liver, pancreas and islet cells and lung, reinforcing their value as potential therapeutics.
Another study by the group (in the hairless mouse skin model) showed that siRNA-based SNAs penetrate the epidermal barrier of the skin. In subsequent studies, the team demonstrated that, when SNAs are applied topically using commercial moisturizers, they penetrate deep within the skin and can be used as a gene regulation technology to selectively target disease-causing genes. Once in the cells, the SNAs simply "flip the switch" of the disease-causing genes to "off," Mirkin explains. Targets for the new therapy include melanoma, squamous cell carcinoma, psoriasis and diabetic wound healing, among others.
"Thanks to the Human Genome Project and all of the genomics research over the last two decades, we have an enormous number of known targets," Mirkin says. "We can use the same tool—SNAs—for each, simply by changing the sequence to match the target gene. What we're seeing is a whole new platform emerging that will enable nucleic acid-based therapies in a way we dreamed about a decade ago, but are just now beginning to realize."
April 8, 2013