BME PhD Prospectus Defense - Xirui Zhang

3:00 pm on Tuesday, October 30, 2012
5:00 pm on Tuesday, October 30, 2012
PHO 339
Title: “An improved high-throughput biosensor platform for in vitro detection and quantification of DNA-protein interactions”

Advisor: Selim Ünlü, ECE/BME
Chair: Selim Ünlü, ECE/BME
Michael Smith, BME
Bennett Goldberg, Physics
Charles DeLisi, Bioinformatics/BME

The functions of genomic DNA are realized through critical interactions with DNA-binding proteins, such as in DNA replication, transcription, and repair. The intrinsic variant conformations of DNA are recognized, stabilized, or even enhanced upon the formation of DNA-protein complexes. These conformational changes of DNA play an important role in specific recognition of DNA sequences by proteins, such as in gene expression and maintenance. Thus, understanding the intrinsic and deformed conformations of DNA’s structure in specific DNA-protein complexes is of considerable biomedical significance. The detection and characterization of specific DNA-protein interactions remain significant challenges because critical dimensions of molecular interactions are on sub-nanometer scales. Existing approaches, such as X-ray crystallography, EMSA, AFM, NMR, and even FRET, are indirect, inherently cumbersome, provide limited information about the binding location, and do not provide the high-throughput capability to investigate sequence dependence of protein-induced DNA conformational changes.

This prospectus aims to develop a high-throughput and user-friendly biosensor platform for precise quantified measurement of conformations of DNA in its native state and in DNA-protein complexes. A dual-color Spectral Self-interference Microscopy (SSFM) will be developed to quantify the conformations of surface immobilized oligonucleotides by precise co-localization of two axial positions. High-speed label-free quantification will also be integrated into the system to perform measurements of biomass accumulation in parallel. The following aims are proposed to demonstrate the versatility of our biosensor platform as a research tool and facilitate impactful discoveries in the biomedical field. First, the tunable property of a smart charged polymer scaffold will be investigated and used to quantify intrinsic conformations of dsDNA with local secondary structures. Then, the effect of sequence variability on the structural changes of dsDNA in DNA-protein complexes will be studied with dynamic measurements of protein binding. The protein Integration Host Factor (IHF) will be used as the model for our platform. The hypothesis is that non-specific DNA-protein interactions can also have high affinity but cannot induce specific conformational changes. The findings from this study will shed light on the mechanism of specific recognitions of proteins to DNA. Finally, we will endeavor to detect conformational distortions of dsDNA with nicks and nucleotide gaps and the repair of these DNA lesions in vitro in physiological conditions through direct observation of DNA conformational changes and protein binding events. Monitoring DNA repair is also an example of applying the biosensor platform to study more challenging processes of DNA-protein interactions. Successful detection of DNA repair in real-time will further lead the biosensor into a screening tool for discoveries of DNA repair inhibitors in cancer treatments.