Aditi Vasudevan's HRP Aptamer Project (2017)

Aptamer against Horseradish Peroxidase (HRP) in Cancer Detection

Introduction

Researchers have been looking into ways of early detection of pre-cancerous and cancerous cells in the breast. Carriers of the BRCA1 and BRCA2 gene mutations are at a higher risk of developing the disease. Frameshift, nonsense, and splicing mutations in these genes produce truncated proteins, which can cause breast and ovarian cancers (Mehrgou 2016). Therefore, the detection of the specific genes would be very useful in the early detection of breast cancer and would help reduce the many invasive cases and deaths caused by this disease. However, the detection of these genes is difficult due to the gene’s size, frequent mutations, and high costs of detection (Mehrgou 2016). In this study, aptamers bound to the target, Horseradish Peroxidase, are being selected to be used as a recognition tool for a cancerous gene. An aptamer bound to the HRP target would be a cost effective and efficient route in the detection of the BRCA1 and BRCA2 mutated genes in breast cancer.

Horseradish peroxidase, or HRP, is an enzyme that has various diagnostic capabilities due to its production of a color indicator. HRP, in the presence of ABTS, OPD, or TMB, catalyzes the oxidation of these substrates and produces a colored product. The enzyme functions most efficiently at near-neutral pH, has a molecular weight of 40 kDa, and is a monomer (Thermo Fisher Scientific). Figure 3 shows a three-dimensional representation of the Horseradish Peroxidase enzyme (Al-Fartusie). HRP has been used as a reporter enzyme in several cases such as detecting HIV-1 envelope peptides and the quantification of protein kinase activity (Krainer and Glieder 2015).

An aptamer can be defined as molecules and oligonucleotides that bind to specific targets with high affinity. The SELEX method for aptamer selection has been proven to be useful in discovering DNA and RNA aptamers (Qinchang 2015). Figure 1 shows the SELEX method, detailing the methods of collection, incubation, and amplification (Barman 2015). In more detail, the SELEX procedure includes 8 distinct steps. In the protein binding step, the magnetic beads bind with the HRP protein using the biotin/streptavidin complex. The procedure further involves successive washes, selection of the potential aptamers to bind to targets, reverse transcription, cycle course/large scale transcription for amplification, transcription, PAGE, purification of the full-length RNA product, RNA quantification, and Binding assay/Sequencing, to check if an aptamer was found. In terms of diagnostics, aptamers play a similar role to antibodies, however, they are easier and cheaper to manufacture and mass produce.

In this study, the aptamer will serve as a diagnostic tool as it would detect the presence of mRNA biomarkers for breast cancer. The basis of detection is that the aptamer will inhibit the horseradish peroxidase enzyme, allowing no reaction to take place when the enzyme is in the presence of its substrate. However, if the aptamer becomes disfigured (due to its binding with another molecule), the horseradish peroxidase active site would be open, allowing for HRP to interact with its substrate and produce a colored product. So once the aptamer has been found, a RNA sequence, complementary to the mutated BRCA1 or BRCA2 genes, will be synthesized onto the aptamer. When the synthesized sequence, attached to the aptamer, binds to the mutated BRCA1/BRCA2 mRNA, the aptamer will disfigure and unattach from the HRP molecule. When this occurs, the HRP enzyme is free to interact with its substrate, ABTS, OPD, or TMB, and produce a colored product. In summary, when the aptamer keeps it structure and stays bound to HRP, there is no response. However, when aptamer binds to the mutated mRNA through complementary base pairing, the aptamer loses its structure, and the HRP protein is free to react with its substrate and produce a visual response. Figure 2 illustrates this process. A potential application of the aptamer could be in an in vitro assay for cancer detection. The mRNA would be isolated from cells like those in blood and the detection of mutated genes would be done outside the body. If the mRNA in the blood sample contained the BRCA1 and BRCA2 mutations, it would interact with the aptamer, and a colored product would be produced.

Currently, one main method of detection of breast cancer are mammograms, physical exams, and self-examinations. Although mammograms prove to be useful, it is only a feasible option once the breast cancer has developed and potentially become invasive. Detecting the presence of the mutated BRCA genes would help in the early diagnosis of breast cancer, even before visual symptoms began. Women with this gene would be able to take more caution and be more aware of the changes in their bodies. The cancer would be identified at its earliest stage, before it could have the chance of becoming invasive and leading to death.

Round 1 of aptamer selection is currently in progress. Last semester, target immobilization, the successive washes, reverse transcription, and PCR (Polymerase Chain Reaction) were performed. However, no bands were visible and there was some contamination in the NTC. After performing three control checks for contamination, it was confirmed that there was contamination somewhere in the process. This semester, round one is currently in progress again and has been completed up to ccPCR. After many rounds of aptamer selection of the HRP target against the N50 RNA pool, the potential aptamers would be sent for Sanger sequencing and a binding assay would be performed to detect whether an actual aptamer has been obtained.

Click Here for Final Report

References

Al-Fartusie, F.S. "Engineering and Characterisation of Novel Protein Covalent Linkages in Horseradish Peroxidase (Hrp): Effect on Structure and Function." Ph D Thesis. N.p., n.d. Web. 09 Apr. 2017. .

“Breast Cancer.” American Cancer Society, www.cancer.org/cancer/breast-cancer.html.

Bonifert, Günther et al. “Recombinant Horseradish Peroxidase Variants for Targeted Cancer Treatment.” Cancer Medicine 5.6 (2016): 1194–1203. PMC. Web. 9 Apr. 2017.

"IHC Immunodetection." Thermo Fisher Scientific. N.p., n.d. Web. 09 Apr. 2017. .

Krainer, Florian W., and Anton Glieder. “An Updated View on Horseradish Peroxidases: Recombinant Production and Biotechnological Applications.” Applied Microbiology and Biotechnology 99 (2015): 1611–1625. PMC. Web. 9 Apr. 2017.

Lisanti, M. P., Martinez-Outschoorn, U. E., Lin, Z., Pavlides, S., Whitaker-Menezes, D., Pestell, R. G., … Sotgia, F. (2011). Hydrogen peroxide fuels aging, inflammation, cancer metabolism and metastasis: The seed and soil also needs “fertilizer.” Cell Cycle, 10(15), 2440–2449. http://doi.org/10.4161/cc.10.15.16870

Mehrgou, A., & Akouchekian, M. (2016). The importance of BRCA1 and BRCA2 genes mutations in breast cancer development. Medical Journal of the Islamic Republic of Iran, 30, 369.

Prediger, Ellen. “Planning to Work with Aptamers?” Planning to Work with Aptamers?, Integrated DNA Technologies, www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2016/03/15/planning-to-work-w ith-aptamers.

Sager, Ruth. “Expression Genetics in Cancer: Shifting the Focus from DNA to RNA.” Proceedings of the National Academy of Sciences of the United States of America 94.3 (1997): 952–955. Print.

Veitch, Nigel C. "Horseradish Peroxidase: A Modern View of a Classic Enzyme." Phytochemistry 65.3 (2004): 249-59. Web.

Why is early diagnosis important? (2017, April 06). Retrieved October 09, 2017, from http://www.cancerresearchuk.org/about-cancer/cancer-symptoms/why-is-early-diagnosis-important

Zhu, Qinchang, Ge Liu, and Masaaki Kai. "DNA Aptamers in the Diagnosis and Treatment of Human Diseases." Molecules 20.12 (2015): 20979-0997. Web