Description:
OBJECTIVE: To build artificial antibodies using DNA origami and develop novel types of electo-optical based biological sensing methodologies. DESCRIPTION: Nature is adept at producing molecules that can recognize and specifically bind to other molecules. In biological systems, antibodies can search out and selectively bind to specific target molecules in the presence of numerous other substances. Antibodies are a critical component of the immune systems of many organisms. Selective binding allows the body"s immune system to target and eliminate specific antigens. Antibodies have also become the gold standard for many biosensing applications. Natural proteins are widely used in diagnostic tests for many diseases because they recognize and efficiently bind to disease markers. Attempts have been made to synthesize molecules with abilities for selective bonding that are comparable to antibodies. However, protein antibodies are difficult and expensive to synthesize in the laboratory. The precise rules of protein folding are still a mystery and are one of the unsolved problems of modern biology. Attempts to manufacture proteins with predetermined shapes and functionality have met with limited success. Protein antibodies are also perishable and possess a short shelf life. Attempts have been made to manufacture antibodies from polymers using molecular imprinting. In molecular imprinting, a solution containing a polymer grows around a target molecule. The target molecule is then washed away. When the molecular imprinted antibody contacts the target molecule, binding occurs. Molecular imprinted antibodies have had some success, However, in the case of biological warfare agents, the use of antigens during the manufacturing process presents a difficult, if not impossible, situation. The folding of single- and double-stranded DNA is a chemically well-understood and controllable process. DNA is generally associated with the storage of genetic information. However, in many ways, it is also an ideal building material. DNA"s sequence dictates its shape and structure. Recently, progress has facilitated cheap and easy manufacturing of DNA strands with custom sequences. The use of self-assembled DNA sequences is thus a very attractive approach in the search for artificial antibodies. The science of DNA origami has recently progressed to the point that it is now possible to design and manufacture complex structures using DNA folding techniques. Much of the science of DNA origami is centered on producing better design software. The ability to produce better design software is a critical component of the controlled design and production large complex structures using DNA origami. PHASE I: Conduct a feasibility study of producing artificial antibodies using DNA origami. Methods should be developed to design structures with predetermined shapes and functions. Methods for manipulating the physical properties of the self-assembled structures should also be examined, and electro-optical testing methods should be defined for verifying the types of physical properties achieved. In particular, methods should be developed to manipulate the charge on the surface of a DNA self-assembled nanostructure. Also, the production of controlled hydrophobic and hydrophilic surfaces should be examined using DNA origami methods. A preliminary design should be made to produce structures using DNA origami that will function as antibodies. Where possible, target antigens related to biological warfare agents or simulants should be used in the initial design studies. PHASE II: Fabricate artificial antibodies using DNA origami. Test the affinity of the antibodies to known antigens. Based on the results of the tests, refine the design of the antibodies. Examine methods for incorporating the new artificial antibodies into sensors specifically designed for the detection and identification of biological warfare agents and simulants. Here, an emphasis should be placed upon defining and refining electro-optical based transduction methodologies for achieving the detection and identification capability. PHASE III DUAL USE APPLICATIONS: Further research and development during Phase III efforts will be directed toward refining final deployable designs for artificial antibodies. Design modifications based on results from tests conducted during Phase II will be incorporated. Manufacturability specific to U.S. Army CONOPS and end-user requirements should be examined. Artificial antibodies will have numerous commercial applications, particularly in the field of medicine. It is expected that commercialization will accelerate once the antibodies become less expensive and easier to use. It is one of the goals of this effort to produce affordable, stable antibodies that can be reliably mass-produced for battlefield applications, especially in the context of biological agent detection and identification. REFERENCES: 1. Gnter Wulff,"Molecular Imprinting in Cross-Linked Materials with the Aid of Molecular Templates - A Way towards Artificial Antibodies", Angewandte Chemie International Edition, volume 34, issue 17, pages 1812-1832, 2003. 2. Yoshitaka Iba and Yoshikazu Kurosawa,"Comparison of strategies for the construction of libraries of artificial antibodies", Immunology and Cell Biology, volume 75, pages 217-221; 1997. 3. Carl A. K. Borrebaeck,"Antibodies in diagnostics from immunoassays to protein chips", Immunology Today, volume 21, issue 8, pages 379-382, 2000. 4. Paul W. K. Rothemund,"Folding DNA to create nanoscale shapes and patterns", Nature, volume 440, number 7082, pages 297-302, 2006. 5. Anton Kuzyk, Bernard Yurke, J. Jussi Toppari, Veikko Linko, and Pivi Trm,"Dielectrophoretic Trapping of DNA Origami", SMALL, volume 4, number 4, pages 447-450, 2008. 6. James C. Mitchell, J. Robin Harris, Jonathan Malo, Jonathan Bath, and Andrew J. Turberfield,"Self-Assembly of Chiral DNA Nanotubes", Journal of the American Chemical Society, volume 126, number 50, pages 16342-16343, 2004. 7. Faisal A. Aldaye, Alison L. Palmer, and Hanadi F. Sleiman,"Assembling Materials with DNA as the Guide", Science, volume 321, number 5897, pages 1795-1799, 2008. 8. Ebbe S. Andersen, Mingdong Dong, Morten M. Nielsen, Kasper Jahn, Ramesh Subramani, Wael Mamdouh, Monika M. Golas, Bjoern Sander, Holger Stark, Cristiano L. P. Oliveira, Jan Skov Pedersen, Victoria Birkedal, Flemming Besenbacher, Kurt V. Gothelf, and Jrgen Kjems,"Self-assembly of a nanoscale DNA box with a controllable lid", Nature, volume 459, pages 73-76, 2009. 9. Rahul Chhabra, Jaswinder Sharma, Yonggang Ke, Yan Liu, Sherri Rinker, Stuart Lindsay, and Hao Yan,"Spatially Addressable Multiprotein Nanoarrays Templated by Aptamer-Tagged DNA Nanoarchitectures", Journal of the American Chemical Society, volume 129, number 34, pages 10304-10305, 2007. 10. Nadrian C. Seeman,"An Overview of Structural DNA Nanotechnology", Molecular Biotechnology, volume 37, number 3, pages 246-257, 2007. 11. W. Shen, H. Zhong, D. Neff, M. L. Norton,"NTA directed protein nanopatterning on DNA Origami nanoconstructs", Journal of the American Chemical Society, volume 131, number 19, pages 6660-6661, 2009. 12. Constantin Pistol and Chris Dwyer,"Scalable, low-cost, hierarchical assembly of programmable DNA nanostructures", Nanotechnology, volume 18, number 12, pages 125305/1-125305/4, 2007.
