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Platform for Quantified High-throughput Measurement of Protein Induced Conformation Changes in DNA

Members: M. Selim Ünlü

 

Alumni: Sunmin Ahn, George Gaby Daaboul, Joseph Thomas Greenspun, Margo R. Monroe, Philipp Stefan Spuhler

 

Researchers at Boston University’s College of Engineering, the Technical University of Munich (TUM) and Nanyang Technological University in Singapore have demonstrated a new strategy to more efficiently measure protein-induced DNA shape, or conformational changes in real-time. Conceived by Professor Selim Ünlü and implemented by Philipp Spuhler, a graduate student in Ünlü’s lab, the new strategy exploits a platform developed at TUM that can accommodate multiple spots of microarrayed dsDNA on individually controlled, lithographically-designed electrodes.

Ultimately, scientists could use the platform to systematically investigate the interactions between selected proteins and hundreds of thousands of different DNA sequences in a single experiment. In the Jan. 4 early edition of the Proceedings of the National Academy of Sciences, the researchers reported on their first experiments with the TUM platform.

“Our results demonstrate the ability to discern between different conformational changes in dsDNA due to sequence-specific binding with a protein,” said Ünlü, the study’s principal investigator. “We believe this work is the first clear demonstration of a viable method for measurement of protein-induced conformational changes in immobilized dsDNA.”

To measure these changes in real time, the researchers anchored fluorescent-labeled DNA molecules to electrodes on the gold surface of the TUM platform, and applied an electric field to orient the fluorophores in a well-ordered manner conducive to effective detection of conformational changes. They next introduced a DNA-binding protein and measured its impact on the intensity of the fluorophores. Since this intensity increases with height above the gold surface, the researchers could deduce any resulting height changes in segments of the DNA molecules—and thus pinpoint corresponding changes in their shape. “When you introduce a protein in solution, the protein will bind to the DNA and change its shape, resulting in a change in the spectral signature we observe from the fluorescent molecules attached to the DNA,” said Ünlü. “We can then detect the spectral signature change and attribute that to a conformational change.”

In the National Science Foundation study, Spuhler introduced a protein called E. Coli Integration Host Factor (IHF) that can bend DNA molecules by as much as 180 degrees, and observed protein-induced bending upon the binding of IHF to the dsDNA probes.

The researchers next plan to combine electrical orientation of fluorescent-tagged DNA with a new optical technique developed by Unlu that uses self-interference fluorescence microscopy (SSFM) to more precisely measure the height of the fluorophores. They aim to eventually use SSFM to analyze different protein-DNA interactions and to identify and characterize specific proteins that induce considerable shape changes in DNA molecules.

DNA with Probes, controlled with Electric Fields

Figure 1: By applying a repulsive electric field at the gold surface to induce a standing orientation in the DNA probes, scientists can more easily discern and measure changes in their conformation due to IHF protein binding. (Image courtesy of PNAS.)

Publications

P. S. Spuhler, L. Sola, M. R. Monroe, M. Chiari, and M. S. Ünlü, "Measurement of potential dependent DNA orientation on indium tin oxide surfaces by fluorescent-self interference microscopy," 12th International Conference on Transparent Optical Networks, Munich, June 2010

J. Knezevic, P. S. Spuhler, Q. Bao, P. Dröge, M. S. Ünlü, and U. Rant, "A platform for in situ real-time measurement of protein induced conformational changes of DNA," Biosensors, 2010, Glasgow, Scotland , May 2010

P. S. Spuhler, J. Knezevic, A. Yalcin, Q. Bao, E. Pringsheim, P. Dröge, U. Rant, and M. S. Ünlü, "Platform for in situ real-time measurement of protein-induced conformational changes of DNA ," Proceedings of the National Academy of Science, 4 January 2010

P. S. Spuhler, J. Knezevic, A. Yalcin, Q. Bao, P. Dröge, U. Rant, and M. S. Ünlü, "A Platform for in situ Real-Time Measurement of Protein Induced Conformational Changes of DNA," Proceedings of IEEE Photonics Society 2009 Annual Meeting, 7 October 2009

P. S. Spuhler, J. Knezevic, A. Yalcin, Q. Bao, P. Dröge, U. Rant, and M. S. Ünlü, "Real-Time Kinetics Measurements of Protein Induced Conformational Changes in DNA," Proceedings of IEEE Photonics Society 2009 Annual Meeting, 7 October 2009


Support

National Science Foundation


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