The breast cancer susceptibility protein, BRCA1, is a tumor suppressor that helps maintain genomic integrity. Changes in BRCA1 that effect DNA repair processes can fuel cancer induction. The Kelly lab, at the Virginia Tech Carilion Research Institute, has recently developed a new methodology that employs silicon nitride (SiN) microchips to isolate BRCA1 assemblies from the nuclear material of breast cancer cells. These microchips are coated with adaptor proteins that include antibodies against target proteins of interest. The adaptor proteins are added in sequential steps to the coated microchips, followed by an aliquot of sample containing the protein of interest, such as BRCA1. The Kelly lab, partnered with Protochips Inc., developed these devices as a robust, tunable platform to monitor molecular processes, and refer to them as 'Cryo-SiN' in cryo-Electron Microscopy (EM) imaging. We are currently using Cryo-SiN to recruit BRCA1 protein assemblies to the microchip surface under mild conditions, while simultaneously preparing them for cryogenic preservation and EM imaging. This strategy presents a viable alternative to antibody affinity columns that require stringent elution steps to obtain protein complexes from the column. Another advantage of the microchip strategy is that it requires only a 30-minute nuclear extraction, a 60-minute enrichment procedure, and a 5-minute microchip capture step--a total of 95 minutes from initially lysing the cells to plunge-freezing the EM specimens. Therefore, these novel approaches represent a major departure from classical separation procedures that often require days to complete, during which time active protein assemblies can readily dissociate or become inactive. Overall, our use of BRCA1-specific microchips may reveal changes in the BRCA1 architecture during various stages of cancer progression--a major gap in knowledge that persists in cancer research. / M. S. / Modern advances in the imaging technology used for cryogenic electron microscopy (cryo-EM) have offered researchers an extraordinary view into the world of biology at the nanoscale. Supplemental to these technical innovations is the development of tunable substrates based on functional new materials that revolutionize the sequestering of biological components from human cells, such as protein complexes formed in breast cancer cells. New developments of novel viewing substrates, given traditional electron microscopy viewing grids have remained unchanged for decades, is the logical next step into the future of enhanced cryo-EM imaging. Tunable microchip substrates, made using recently enhanced micro-engineering techniques, are currently under development for use in cryo-EM imaging. In this work I have examined these microchip substrates for their capacity to streamline the isolation of biomolecules such as the protein most prominently cited in breast cancer, known as the breast cancer susceptibility protein (BRCA1). Utilizing these novel microchip substrates in the Kelly Lab, I have collected and analyzed data containing BRCA1 proteins, formed in human breast cancer cells, toward the development of 3-dimensional protein structures that allow us to peer into the structure-function relationships of these proteins. New and exciting Cryo-EM data, collected using these newly developed microchips, has the potential to reveal obscure disease mechanisms being propagated at the molecular level in modern clinical practice, such as breast cancer.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/94418 |
| Date | 16 April 2018 |
| Creators | Alden, Nicholas Andrew |
| Contributors | Mechanical Engineering, Kelly, Deborah F., Behkam, Bahareh, Nain, Amrinder |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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