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Researchers Develop Molecular Toolkit to Observe BRCA1 Proteins

NEW YORK (GenomeWeb) — Scientists from Virginia Tech's Carilion Research Institute have created a molecular toolkit in order to visualize and structurally analyze the BRCA1 protein and its associated parts in a near-native environment. 

"[Using this technology] we were able to make models about how this suspicious region doesn’t work properly [in mutated BRCA1 genes]," said Deborah Kelly, lead author and assistant professor of biological sciences in Virginia Tech’s College of Science, in an interview. 

Describing their method in a recently published article in Nature Scientific Reports, the scientists created enriched portions of RNA Polymerase II that contained BRCA1 and BARD1 proteins, and applied them to a set of silicon nitride microchips. The microchip surface was coated with antibodies raised against the BRCA1 protein, and once the proteins were collected by the microchips they were negatively stained.  

All microchips were then inserted into a FEI Spirit BioTwin Transmission electron microscope. Then the scientists used automated routines in the PARTICLE software package and selected images with full BRCA1-associated RNA Polymerase II core complexes from approximately 22,000 individual images taken from the microchip. Selected BRCA1-transcriptional assemblies were exported as an image stack into the RELION software package, and based on the statistical likelihood comparisons of the images with their RNAP II model they found five distinct structures for the protein assemblies. They then determined relative location of the BRCA1 N- and C- terminals by analyzing the structures using the SPIDER software package

BRCA1 is a tumor suppressing protein and helps proofread the DNA templates and works to correct errors, which means it plays a role in ensuring the stability of the cell's genetic material, according to the National Cancer Institute. When mutations occur in BRCA1, normal cellular processes that regulate proper growth and division are disrupted — and these disruptions are heavily correlated with breast and ovarian cancers. 

Understanding more about BRCA1's structural function in mutated and normally functioning genes could help scientists develop better and more focused treatments. 

Kelly and her team were able to get a fuller picture of transcriptional processes happening between BRCA1 proteins, BARD1 proteins, and DNA throughout different regions of the RNAP II core complex. The method opens the door to further research on these types of processes that drive cells to a pathological state. 

A key advantage of using the microchip capture system is that it takes approximately 95 minutes from lysing the cells to preparing the specimens, according to the researchers. It's a major departure from classical specimen preparation procedures involving labeling, staining, and enrichment that often require days to complete. 

Another advantage of the technology is that it has the potential to be very versatile, Kelly said, noting that the researchers had used the chips to analyze stem cells to try and determine their role in neurodegeneration.  "I definitely see its use in a lot of cancers and even different types of disease pathogen," she said. 

The technology offers the opportunity to get new insight into the structural interactions between different proteins and cells that can unravel some of the lesser understood mechanisms, as well as the possibility to create better disease models, the researchers believe. 

While Kelly thinks the technology has a great deal to offer, since the base microchips are commercially available and the procedures are available through the scientific literature, her lab isn't planning to commercialize the toolkit, she told GenomeWeb. Although, she did indicate that she and her colleagues would be happy to help facilitate the use of the technology for future research and are open to other scientists reaching out to them on its use.