Jenny.Green@asu.edu
MaryZhu @asu.edu
Chemistry.asu.edu

• Spring 2011
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The licensed technologies include specialized approaches for DNA base sensing and reading and build on an ongoing collaboration between Roche's sequencing center of excellence, 454 Life Sciences, and IBM to develop and commercialize a single-molecule, nanopore DNA sequencer with the capacity to rapidly decode an individual's complete genome for well below $1000.

The licensed technologies offer novel approaches for reading the sequence of bases, or letters, in a single DNA molecule as it is passed through a nanopore. The team has demonstrated proof-of-concept, and is in the midst of making a third generation reader molecule that provides better discrimination between the DNA bases. The licensing agreement with Roche will help translate these discoveries into a commercial instrument.

"Our promising approach, which combines advances in physics, electronics and nanotechnology, eliminates the need for the use of a major cost of sequencing today – namely, the use of chemical reagents – to read an individual's genome," said Lindsay, an ASU Regents' Professor and director of the Biodesign Institute's Center for Single Molecule Biophysics.

The DNA Transistor technology, developed by IBM Research, slows and controls the movement of the DNA molecule as it threads through a microscopic nanopore in a silicon chip, while the newly licensed DNA reading technology can decode the bases of the DNA molecule as it passes through. Both technologies are centered on semiconductor-based nanopores, which have advantages over protein-based nanopores in terms of control, robustness, scalability, and manufacturability.

The deal was brokered by Arizona Technology Enterprises (AzTE), the exclusive intellectual property management and technology transfer organization of Arizona State University, and includes sponsored research funding that will help Lindsay's team move the technology towards commercialization. The National Human Genome Research Institute (NHGRI), part of the National Institutes of Health (NIH), recently awarded Lindsay and fellow Biodesign researcher Bharath Takulapalli more than $5 million for their work in DNA sequencing. ASU was the only university to receive more than one award.

Antibodies are the backbone of the immune system - capable of targeting proteins associated with infection and disease. They are also vital tools for biomedical research, the development of diagnostic tests and for new therapeutic remedies. Producing antibodies suitable for research however, has often been a difficult, costly and laborious undertaking.

Now, John Chaput and his colleagues at the Biodesign Institute at Arizona State University have developed a new way of producing antibody-like binding agents and rapidly optimizing their affinity for their target proteins. Such capture reagents are vital for revealing the subtleties of protein function, and may pave the way for improved methods of detecting and treating a broad range of diseases. The team�s results appear in today�s issue of the journal ChemBioChem.

Antibodies are Y-shaped structures, capable of binding in two or more places with specific target proteins. Synthetic antibodies are much simpler forms that attempt to mimic this behavior. As Chaput explains, creating affinity reagents with strong binding properties can be accomplished by combining two weak affinity segments on a synthetic scaffold. The resulting affinity reagent, if properly constructed, can amplify the binding properties of the individual segments by two or three orders of magnitude.

"This dramatic change in affinity has the ability to transform ordinary molecules into a high affinity synthetic antibody," Chaput says. "Unfortunately, the chemistry used to make these reagents can be quite challenging and often requires a lot of trial-and-error. With NIH funding, my group has reduced the complexity of this problem to simple chemistry that is user friendly and easily amenable to high throughput automation. Such technology is absolutely necessary if we want to compete with traditional monoclonal antibody technology. " Traditionally, antibodies for research have been extracted from animals induced to produce them in response to various protein antigens. While the technique has been invaluable to medical science, obtaining antibodies in this way is a cumbersome and costly endeavor. Instead, Chaput and his team produce synthetic antibodies that do not require cell culture, in vitro selection or the application of complex chemistry. They call their reagents DNA synbodies.

The new strategy-referred to as LINC (for Ligand Interaction by Nucleotide Conjugates) uses DNA as a programmable scaffold to determine the optimal distance needed to transform two weak affinity binding segments or ligands into a single high affinity protein capture reagent. The result is an artificial antibody, capable of binding to its antigen target with both high affinity and high specificity. The process is rapid and inexpensive. It also offers considerable flexibility, as the distance between the two ligand components bonded to the short, double-stranded DNA scaffold can be fine-tuned for optimum affinity.

In earlier work, the group identified ligand candidates by producing thousands of random sequence peptide chains-strings of amino acids, connected like pearls on a necklace. The peptide sequences were affixed to a glass microarray slide and screened against a target protein to pinpoint those that were capable of recognizing distinct protein binding sites. Two promising ligand candidates could then be combined to form a DNA synbody.

In the current study, the group instead makes use of pre-existing ligands with documented affinity for various disease-related proteins. The method involves the use of well-characterized ligands as building components for high quality DNA synbodies, eliminating the initial screening procedure and expanding the potential to tinker with the two-piece synbody in order to optimize affinity. The peptides of choice for the study were those with high affinity for something called growth factor receptor bound protein 2 (Grb2). Grb2 has many cell-signaling functions and is an important focus of research due to its association with cellular pathways involved in tumor growth and metastasis.

By scouring the scientific literature, the group identified two peptides that recognize distinct sites on the surface of Grb2. Chaput points out, "this is a nice example where a few hours in the library can save you weeks in the lab." The next step was to create an assortment of synbody constructs based on these peptides. To do this, one peptide was attached to the end of a short DNA strand, while the other peptide was attached to the complementary DNA strand further along its length (see figure 1).

Figure 1. Two peptide chains are attached to a segment of double-stranded DNA, displaced by a distance which can be modified to improve binding affinity with a target protein (seen in blue).

The two peptide strands could be attached to the scaffold in either a forward or reverse direction and could be interchanged, with either occupying the terminal end of the first DNA strand. Further, the distance between peptide segments along the DNA strands could be adjusted to yield the best target affinity.

Experiments examined binding affinity for peptide chains separated by 3, 6, 9, 12, 15 and 18 base pairs along the DNA strand, (a distance range of 1.0-6.1 nm). Inspection revealed the best results for a synbody constructed of peptides separated by 12 base pairs at a distance of 4.1 nm, compared with the other 5 constructs.

The results for the best synbody in the study were impressive, demonstrating a binding affinity five- to ten-fold stronger than commercially available antibodies for Grb2, despite the synbody�s comparatively primitive architecture. In further tests, the synbody was shown to exhibit high specificity-isolating Grb2 from other proteins in a complex biological mixture and selectively binding with its target.

The technique offers a new approach to producing high quality affinity reagents for disease research, diagnostic testing and the development of effective therapeutics.

Written by: Richard Harth Science
Writer: The Biodesign Institute
richard.harth@asu.edu

Joshua LaBaer, M.D., Ph.D., of the Biodesign Institute at Arizona State University, has been selected as one of three finalists for the Phoenix Business Journal's Health Care Hero award in the researcher/innovator category. The annual award honors exemplary performance in the Phoenix health care industry. Award recipients will be announced at a breakfast at the Arizona Biltmore Resort on Aug. 18.

Two years ago, LaBaer, a medical oncologist and research scientist, was recruited from Harvard University to serve as the first Piper Chair in Personalized Medicine at ASU's Biodesign Institute. LaBaer leads a multidisciplinary team in a large-scale effort to discover and validate unique molecular fingerprints of disease. These fingerprints, called biomarkers, can provide an early warning for those at risk of major illnesses. LaBaer is co-inventor of a technology that serves as the molecular toolkit for his approach.

LaBaer's recruit to ASU was a boon to the medical and research community, as he is one of the foremost investigators advancing personalized medicine. He is working in a new field of science called proteomics to make major strides in cancer research, as well as diabetes and infectious diseases.

Recently, LaBaer identified a panel of 28 new biomarkers that may aid in the early diagnosis of breast cancer. These biomarkers are important in breast cancer tumor biology and pathology, as well as drug resistance to leading treatments.

"We believe that these biomarkers will lead to the first type of blood tests for the early detection of breast cancer," said LaBaer. "We hope this will lead to catching breast cancer earlier and saving thousands of lives each year."

In addition to early detection, breast cancer care at present falters when patients become resistant to the drugs used to treat the disease. LaBaer's group recently uncovered 30 novel genes involved in tamoxifen drug resistance. They did this by screening genes implicated in the development of breast cancer cells that become resistant to the drug with a robotic system that places human genes into cancer cells to determine if the genes would change the behavior of those cells.

LaBaer's center is home to a unique, worldwide resource for research called DNASU, a gene library of more than 127,000 unique genes made in his lab and contributed from labs elsewhere. These DNA samples represent more than 750 organisms and provide the raw materials for experiments. His lab has made this library into a shared resource to accelerate research. Housed in a million-dollar custom robotic freezer, the gene library is the only one of its kind in the southwestern United State. To date, they have distributed 250,000 plasmid clones to 550 labs in 35 countries.

In addition to his innovations, LaBaer's community impact has been significant. His center has support from the Breast Cancer Research Foundations, the National Cancer Institute, the National Institute for General Medical Sciences, the National Institute for Allergy and Infectious Diseases, and a $35 million philanthropic gift from the Piper Charitable Trust. LaBaer's center has resulted in jobs, opportunities for students, incoming grant dollars, plus the attention that his cutting-edge research brings.

LaBaer earned a BS from UC-Berkeley, and his MD and PhD degrees at UCSF. He is a board certified physician in Internal Medicine and Medical Oncology and has published 106 publications.

LaBaer is the founder of the International Human Proteome Organization and US HUPO. He serves on the NCI Board of Scientific Advisors and co-chair for the Steering Committee for NCI's Early Detection Research Network. He also serves as an associate editor for the Journal of Proteome Research.