This research incorporates micro-scale materials engineering, surface chemistry, physics and biochemistry to answer fundamental questions about biology that cannot easily be addressed through traditional experimental methods. The overall goal encompasses a classic problem in biochemistry, namely: How do site- or structure-specific DNA-binding proteins locate their targets among a vast excess of nonspecific DNA? To help address this question, the Greene laboratory is using total internal reflection fluorescence microscopy (TIRFM) as a tool to directly visualize individual protein complexes as they search for their target sites on single molecules of DNA. The Greene laboratory is also developing new methods that will allow the construction of aligned arrays comprised of hundreds of individual DNA molecules, which are suspended above an inert lipid bilayer and organized into patterns with user-defined orientations, tensions, and topologies. These DNA arrays will allow for rapid collection of statistically relevant information from hundreds of individual molecules by making possible parallel processing of multiple reaction trajectories. These novel research tools will be used to determine how proteins that are involved in post-replicative repair of mismatched bases locate and respond to their specific targets. Despite years of intensive investigation these mechanisms remain unknown, largely due to the inherent limitations of traditional ensemble-level biochemical measurements. These new single-molecule approaches can be used to determine exactly what proteins are bound to DNA, where they are bound, how they behave, when they leave, and how they influence one another, all in real-time at the single-molecule level. The technology-driven methods developed during the course of this research will provide a high-throughput approach for single-molecule analysis of nucleoprotein complexes, which can be applied towards the study of virtually any biological system that involves the interactions between protein and DNA molecules. This interdisciplinary work also provides trainees with a cutting-edge, broad-based educational experience that will allow them to successfully contribute to the scientific community upon completion of their degree requirements. To promote the understanding of single-molecule approaches, these emerging technologies will be integrated into the university's graduate course curriculum; several departmental lectures will be scheduled featuring leading experts from around the country; and a regional discussion group/symposium will also be organized to stimulate interactions and communication between the laboratories in the New York area that are interested in single-molecule research. Dr. Greene has initiated a separate project that will be conducted solely by undergraduate and high school students. The goal of these efforts is to incorporate younger students into all aspects of scientific work performed in the laboratory, thereby providing them with valuable, real-world research experience.
Research: The Greene laboratory has developed unique methods for viewing hundreds of aligned DNA molecules in real-time using optical microscopy. In brief, the surface of a microfluidic chamber is coated with a lipid bilayer, and DNA molecules are tethered to the bilayer. The bilayer renders the surface inert and it permits tethered DNAs to move in two dimensions, yet confines them near the flowcell surface. Buffer flow is used align the DNA at nanofabricated diffusion barriers, allowing visualization of hundreds of single DNAs in a field-of-view. DNA curtains provide a powerful experimental platform enabling acquisition of statistically relevant information from hundreds of individual reactions. With these new research tools in hand we are now in a unique position to pursue a number of projects related to protein-DNA interactions, with a common theme of trying to understand fundamental aspects of how proteins move on DNA while searching for specific binding targets. Our NSF funded work has focused on understanding how proteins involved in DNA repair find and respond mismatched bases in DNA. Our findings help establish how different modes of facilitated diffusion can be utilized during the early stages of DNA mismatch repair. Broader Impacts: The technologies developed in our group during the prior NSF funding period are applicable to a range of problems involving biological macromolecules, which has generated substantial interest from the scientific community. Dr. Greene has lectured extensively on the DNA Curtain technology and its applications, including 63 invited seminars outside of Columbia University. Dr. Greene has also mentored students at all stages of their careers in a highly interdisciplinary research setting. The graduate student (Jason Gorman) involved in this work received the 2010 Peter Sajovic Memorial Prize from Columbia University for outstanding work in Biology, the 2011 recipient of the Harold M. Weintraub Graduate Student Award from the Fred Hutchinson Cancer Research Center, and he has begun his postdoctoral research at the NIH. During the prior funding period, Dr. Greene also hosted 7 undergraduates and 3 high school students in his laboratory, and published 5 papers on which undergraduates were listed as co-authors. Publications supported by NSF funding: Gorman, J., Plys, A.J., Visnapuu, Alani, E., and Greene, E.C. 2010. Visualizing 1D-diffusion of eukaryotic DNA repair factors along a chromatin lattice. Nature Structural & Molecular Biology 17(8): 932-938. (Recommended by the Faculty of 1000 Biology: http://f1000biology.com/article/id/4775956/evaluation). Gorman, J., Fazio, T., Wang, F., Wind, S., and Greene, E.C. 2010. Nanofabricated racks of aligned and anchored DNA substrates for single molecule imaging. Langmuir 26(2): 1372-1379. Fazio, T.A., Visnapuu, M-L., Greene, E.C., and Wind, S. 2009. Fabrication of nanoscale curtain rods for DNA curtains using nanoimprint lithography. Journal of Vacuum Science and Technology A 27(6): 3095-3098. Gorman, J., Chowdhury, A., Surtees, J.A., Shimada, J., Reichman, D. R., Alani, E. and Greene, E.C. 2007. Dynamic basis for one-dimensional DNA scanning by the mismatch repair complex Msh2-Msh6. Molecular Cell 28(3): 359-370. (Featured in News & Views, Nature Structural & Molecular Biology Vol. 12, pp. 1124-1125; Rated "Exceptional" by the Faculty of 1000 Biology: http://f1000biology.com/article/id/1095954/evaluation). Visnapuu, M-L., Fazio, T., Wind, S., and Greene, E.C. 2008. Parallel arrays of geometric nanowells for assembling DNA curtains with controlled lateral dispersion. Langmuir 24(19): 11293-11299. Fazio, T., Visnapuu, M-L., Wind, S., and Greene, E.C. 2008. DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging. Langmuir 24(18): 10524-10531. Wang, F., and Greene, E.C. 2011. Single-molecule studies of transcription: from one RNA polymerase at a time to the gene expression profile of a cell. (Review) Journal of Molecular Biology [Epub ahead of print]. Wolcott, H.N., Alcott, B., *Kaplan, L., and Greene, E.C. Nanofabricated DNA Curtains for High-throughput Single-molecule Imaging of Protein-DNA Interactions. In "Microscopy: Science, Technology, Applications and Education". 2010 Edition. A. Méndez-Vilas and J. Díaz (Editors). Volume 4, pages 722-732. *Undergraduate Greene, E.C., Wind, S., Fazio, T., Gorman, J., and Visnapuu, M-L. 2010. DNA curtains for high-throughput single-molecule optical imaging. Methods in Enzymology 472: 293-315. Gorman, J. and Greene, E.C. 2008. Visualizing one-dimensional diffusion of proteins on DNA. (Review). Nature Structural & Molecular Biology 15(8): 768-774. Finkelstein, I.J. and Greene, E.C. 2008. Single molecule studies of homologous recombination. (Review). Molecular Biosystems 4(11): 1094-1104. Visnapuu, M-L., *Duzdevich, D., and Greene, E.C. Investigating Protein-DNA interactions with total internal reflection fluorescence microscopy and DNA curtains. 2007. In "Modern Research and Educational Topics in Microscopy". 2007 Edition. A. Méndez-Vilas and J. Díaz (Editors). Volume 1, pages 297-308. * Undergraduate Patents: Title: "DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging", Inventor: Eric C. Greene, Ph.D.; U.S. Patent Application No.: 61/047,657 Title: "Geometric patterns and lipid bilayer for DNA molecule organization and uses thereof", Inventor: Eric C. Greene, Ph.D.; U.S. Patent Application No.: 61/116,815
