Our long-range goal is to develop a surface array-based detection system to rapidly characterize regulatory molecular interactions. The objectives of this proposal are to develop a high-throughput, double-stranded oligonucleotide array-based DNA binding protein analysis system to identify cis-regulatory DNA binding motifs operative in bacterial and plant cells, and to use matrix-assisted laser-desorption ionization time-offlight mass spectrometry (MALDI-TOF-MS) to identify the interacting proteins. The central hypothesis of this proposal is that specific protein-DNA interactions can be detected by surface plasmon resonance using arrays of 48 different double-stranded DNAs and cell or nuclear extracts. We expect that the hundreds to thousands of different transcription factors present in the extracts will find and bind tightly to their target sequences in a single experiment. We formulated this hypothesis on the basis of our previous work and strong preliminary data. We have demonstrated in our own labs that SPR can sensitively detect specific protein-DNA interactions on surfaces, obviating the need for a labeling step, and we can differentiate between protein and DNA samples spotted in an arrayed pattern. The rationale for the proposed research is that by developing a method to identify cis-regulatory sequences in a high-throughput manner in any cell type, we can make significant gains in our understanding of sequence-driven gene regulation. Such knowledge will be beneficial to all of biology, from informing basic mechanisms in organismic development and responses to the environment, to a clearer view of the diseased state. In addition, all biological processes that contribute to minority health disparities have some uncharacterized component of transcriptional control. The approach will thus be useful in identifying those fundamental regulatory responses that contribute to health disparities in minority communities. We are well prepared to undertake the proposed research, as we have the breadth of expertise in the PI and collaborators to meet our objectives in a conducive, particularly well-equipped research environment.
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