| Summary: | Thesis (Ph.D.)--Boston University === PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. === A deep understanding of disease processes requires understanding of DNA, RNA and protein function on a molecular level. Protein-DNA interactions play a crucial role in many of these processes, including in gene expression. The high-throughput capability of micro-array based technologies enables studies on the sequence dependence to determine the functional roles of protein-DNA binding. In vitro protein binding to double stranded DNA microarrays has proven to be an effective complementary technique to the in vivo methods, as this allows rapid characterization of the protein-DNA binding sequence specificity and much higher resolution of the binding site.
In this dissertation, we apply Spectral Self-Interference Fluorescence Microscopy (SSFM) to develop a platform that improves upon existing in vitro protein binding microarray technologies. The platform permits measurement of the protein binding site without the need for protein tagging with radiolabels or fluorophores. The technology enables precise quantification of DNA conformation changes that are induced by protein binding. There are currently no viable high-throughput techniques available to investigate the sequence dependence on protein induced DNA bending. While they are not well understood, such structural changes are thought to play a critical role in transcription.
A crucial component of the technique is the measurement of DNA orientation, as this permits the detection of the protein binding location and DNA conformation changes. We apply a novel polymeric scaffold to control the surface charge of the sensor and, thus, the orientation of surface immobilized oligonucleotides. The orientation is precisely quantified through sub-nanometer height localization of fluorophore labels on either end of the oligonucleotide probes.
The DNA binding protein Integration Host Factor (IHF) is used as the model system to demonstrate a proof of principle for the platform. IHF is known to bind DNA with high sequence specificity and to induce a 160° bend. The binding position of IHF to the dsDNA probes is resolved to three nucleotides and the conformation changes that result due to a single nucleotide polymorphism in the consensus binding sequence are observed. The platform is scalable to permit observation of protein binding to hundreds of thousands ofunique DNA sequences on a single chip. === 2031-01-01
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