The design and development of the Cognate Site Identifier (CSI) microarray to determine the comprehensive sequence specificity of any DNA-binding molecule

Determining the sequence-recognition properties of DNA-binding proteins and small molecules is critical for understanding how transcription factors regulate gene expression and has important implications in disease and drug development. To address this need, we developed a high-throughput approach, called the Cognate Site Identifier (CSI) microarray, that provides a comprehensive profile of the binding properties of DNA-binding molecules. The CSI microarray displays every permutation of a duplex DNA sequence (up to 12 positional variants) and can be used to obtain the specificity and affinity of biomolecule-DNA interactions.In Chapter 2, we use this platform to determine the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor, both individually and as a cooperative complex on DNA. To minimize the inherent variability of protein-DNA interactions found in early CSI experiments, we describe a seven-step data normalization technique to transform the CSI data into a precise sequence-specificity profile in Chapter 3. Given the comprehensive nature of this CSI profile, we then build upon current data display and analysis techniques via the Specificity Landscape and CSI-Tree in Chapter 4. These two techniques give us unprecedented insights into the sequence recognition properties of the DNA-binding molecules being studied. Finally, in Chapter 5, we combine our comprehensive in vitro CSI approach with the high-throughput in vivo ChIP-chip method to gain a greater understanding than either technique yields individually, of the regulatory behavior of the bacterial transcription factor VFR (Virulence Factor Regulator).To date, we have examined the sequence specificity of numerous small molecules, transcription factors, and cooperative complexes. Most small molecules and transcription factors studied using the CSI microarray have yielded informative DNA-binding profiles, indicating that the binding preferences of almost any DNA-binding ligand can be obtained with this approach. Therefore, we believe the CSI microarray will become a widespread and invaluable technique in the study of any molecule that can interact with DNA.