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Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry
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Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric
Xirui Zhang1, George G Daaboul2, Philipp S Spuhler1
1Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.
Nanoscale
|February 19, 2016
Summary
This study introduces a novel microarray method to simultaneously measure DNA-binding protein amounts and DNA conformational changes. This advance aids in understanding genome maintenance and protein-DNA interactions.
Area of Science:
- Genomics
- Molecular Biology
- Biophysics
Background:
- DNA-binding proteins are essential for genome maintenance and function.
- Understanding protein-DNA binding mechanisms, particularly indirect readout via DNA conformational changes, remains a challenge.
- Current high-throughput DNA microarrays analyze binding affinities but lack conformational data.
Purpose of the Study:
- To develop a quantitative approach for simultaneously measuring protein-DNA binding and DNA conformational changes.
- To integrate this approach into a microarray format for high-throughput analysis.
- To characterize conformational-specific protein-DNA interactions.
Main Methods:
- Utilized spectral interferometry on a layered substrate in two distinct modalities.
- Employed label-free spectral reflectivity to quantify protein and DNA binding amounts.
- Used fluorescence vertical sectioning to measure nanometer-scale DNA conformational changes.
Main Results:
- Successfully demonstrated simultaneous measurement of protein-DNA binding and DNA conformational changes in a microarray format.
- Quantified protein binding to surface-immobilized DNA using spectral reflectivity.
- Measured DNA conformational changes using fluorescence vertical sectioning.
Conclusions:
- The developed approach enables quantitative, large-scale characterization of conformational-specific protein-DNA interactions.
- This method complements existing high-throughput DNA microarray technologies.
- Offers a rapid and simple tool for studying genome-protein dynamics.
