Tunable Microchips for Imaging Protein Structures formed in Breast Cancer Cells

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.

Name thesis:degree_name

M. S.

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Mechanical Engineering

Department dc:contributor.department

Mechanical Engineering

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2018

Author dc:creator

• Alden, Nicholas Andrew

Chair dc:contributor.committeechair

• Kelly, Deborah F.

Committee members dc:contributor.committeemember

• Behkam, Bahareh
• Nain, Amrinder

• Cryo-Electron Microscopy
• Transmission Electron Microscopy
• Silicon Nitride
• Silicon Nitride Microchips
• Molecular Imaging Strategy
• BRCA1
• P53
• Protein Enrichment

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

vt_gsexam:14426

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/94418