Search

The maintenance and functions of the genome are facilitated by DNA-binding proteins, whose specific binding mechanisms are not yet fully understood. Recently, it was discovered that the recognition and capture ofDNA conformational flexibility and deformation by DNA-binding proteins serve as an indirect readout mechanism for specific recognition and facilitate important cellular functions. Various biophysical techniques have been employed to elucidate this conformational specificity of protein-DNA interactions. These techniques are not sufficiently high-throughput to perform systematic investigation ofvarious protein-DNA complexes and their functions. Microarray-based high-throughput methods enable large-scale and comprehensive evaluation of the binding affmities of protein-DNA interactions, but do not provide conformational information. In this dissertation, 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 that determines surface-immobilized DNA conformation by measuring axial height offluorophores tagged to specific nucleotides. The amount of bound protein and DNA are measured by white light reflectance spectroscopy that quantifies molecular surface densities by measuring bioniolecule layer thicknesses. By implementing a dual-spectral imaging configuration, we can perform the two independent interferometric measurements in parallel using two separate spectral bandwidths. [TRUNCATED]