BME PhD Dissertation Defense - Xirui Zhang

10:00 am on Monday, August 18, 2014
44 Cummington Mall, Room 203
Title: "Dual-Spectral Interferometric Sensor for Quantitative Study of Protein-DNA Interactions"

Selim Unlu, Professor, ECE, BME (Advisor)
Michael Smith, Assistant Professor, BME (Chair)
Bennett Goldberg, Professor, Physics
Allison Dennis, Assistant Professor, BME

The maintenance of intact genetic information and functions of the genome are accomplished by DNA-binding proteins, whose specific binding mechanisms are yet fully understood. Recently, it was discovered that the recognition and capture of DNA conformational flexibility and deformation by DNA-binding proteins serve as an indirect readout mechanism for specific recognition. Various biophysical techniques have been employed to elucidate this conformational specificity of protein-DNA interactions, but their throughput is limited by slow and precautious technicalities. High-throughput methods allow for effective and comprehensive analysis of binding specificities by providing large-scale protein-DNA binding data, but do not provide conformational information.

We developed a tool that enables high-throughput quantification of both conformational specificity and binding affinity of protein-DNA interactions. Our approach is to combine quantitative detection of DNA conformational change and protein-DNA binding in a DNA microarray format. The DNA conformational change is measured by spectral self-interference fluorescence microscopy (SSFM) that determines surface-immobilized DNA conformation by measuring axial height of fluorophores tagged to specific nucleotides. The amount of protein binding to DNA is measured by modified white light reflectance spectroscopy that quantifies molecular surface densities by measuring biomolecule layer thicknesses. By designing the DNA microarray substrate and the optical setup, we can perform the two independent interferometric measurements in parallel using two separate spectral ranges.

We used the E. coli integration host factor(IHF) protein as the model system to demonstrate parallel quantification of DNA conformational change and protein-DNA binding. First, we characterized and modeled the conformation of surface-immobilized DNA for the detection of DNA conformational changes. We also proposed a quantitative model to resolve conformational specific binding and nonspecific binding. Based on the model, we evaluated factors affecting protein-DNA interactions on a surface and further demonstrated distinguished and parallel detection of conformational specific and nonspecific IHF-DNA binding. The scalability and versatility of the presented method for studying specific and nonspecific protein-DNA interactions make it a rapid and convenient tool for quantitative and large-scale screening of conformational specific and nonspecific protein-DNA complexes to understand their regulatory functions in the cell.