Katherine Barclay's HRP Aptamer Project (2018)

Aptamer Detection Against Horseradish Peroxidase for Identification of BRCA1 Gene Mutation

Introduction and Background:

Breast cancer is a very common type of cancer among women and has a massive effect on the lives of those diagnosed. Those who get diagnosed endure invasive, expensive, and time intensive treatments but are still at risk for metastasis even if the cancer is temporarily eradicated from the breast tissue. There is a specific gene called BRCA1 on chromosome 17 that, if mutated, can lead to a higher risk of developing cancers like breast cancer. BRCA 1 functions to maintain genomic stability and suppress cell growth (Robertson, 2015). Mutations of this gene also lead to higher probability of developing other cancers like ovarian and cervical cancer (Breast Cancer). It is a major risk factor to inherit a mutated copy of this gene, therefore testing must be done to know if a mutation has taken place in the genome which can be detected by genetic testing that is costly and takes a long period of time. Currently, the cost of testing ranges from $300-$5000 and results take 2-4 weeks to be received (Genetics). With this mutation being an indicator of possible cancer development and test results not being available quickly or being too expensive for the average person, an affordable diagnostic using aptamers is necessitated.

An aptamer that has high specificity for a target could be used in an ELONA (Enzyme Linked Oligonucleotide Assay) detection method of mutant gene product being produced by the mutant BRCA1 gene. An aptamer is an oligonucleotide segment that can be found with a high binding specificity to a target. The target that would be used in this detector is Horseradish Peroxidase (HRP), an enzyme isolated from horseradish roots. HRP can function as a reporter molecule which when oxidized, will show a colorimetric change that can be measured. The aptamer that would bind specifically to HRP will be found using the SELEX (Sequential Evolution of Ligands by Exponential Enrichment) methodology (Figure 2).

This technique involves exposing a pool of RNA to the target (HRP) and seeing what binds, then washing away unbound species. The bound species would then be amplified and purified. Multiple rounds of SELEX would take place to select for a specific, successfully binding aptamer that would be used in an ELONA.

After finding a specific aptamer for HRP using the SELEX method, another aptamer would need to be found that would bind to the mutated protein produced by BRCA1. Both of these aptamers would later be attached to each other and function as a unit to detect mutant protein within a biological sample (Figure 1). Within the ELONA, the aptamer bound to the target would change conformation in the presence of the mutant proteins, releasing Horseradish Peroxidase and allowing it to bind to a substrate molecule within the biological sample and fluoresce. If the mutant tumor suppressor proteins were detected by the aptamer in a diagnostic test, then it would be assumed the BRCA 1 is harmfully mutated. This test would be cheaper and results would be available more rapidly.

Round 1 of aptamer selection to find an aptamer against HRP is being completed. After this aptamer is found, research will continue to effectively construct the ELONA that will be used in the diagnostic tool. This diagnostic tool should provide accurate results showing if the BRCA 1 gene is mutated by indicating presence of mutant protein. This tool will be available to a wider group of individuals who could not have afforded genetic testing and will provide key insight to risk of an individual of developing harmful cancers.

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References:

Apoferritin protein nanoparticles dually labeled with aptamer and horseradish peroxidase as a sensing probe for thrombin detection. (2012, November 17). Retrieved April 02, 2018

Breast Cancer. (2017, June 07). Retrieved March 30, 2018Enzyme Explorer - Peroxidase Enzymes. (n.d.). Retrieved March 30, 2018

European Bioinformatics InstituteProtein Information ResourceSIB Swiss Institute of Bioinformatics. (2018, March 28). Breast cancer type 1 susceptibility protein. Retrieved April 02, 2018

Genetics of Breast and Gynecologic Cancers. (n.d.). Retrieved March 30, 2018

Kuchenbaecker, K. B., Hopper, J. L., Barnes, D. R., Phillips, K. A., Mooij, T. M., Roos-Blom, M. J., . . . Olsson, H. (2017, June 20). Risks of Breast, Ovarian, and Contralateral Breast Cancer for BRCA1 and BRCA2 Mutation Carriers. Retrieved March 30, 2018

Robertson, S. (2015, September 17). BRCA1 Gene Function. Retrieved March 30, 2018