Michael Zamora's HRP Aptamer Project (2016)

Nucleic Acid Aptamer Selection from N71 RNA Pool Against Horseradish Peroxidase (HRP) for ELISA against AMACR protein Implicated in Prostate Adenocarcinoma

Introduction/Background

There is a current need for the development of improved detection methods against various forms of cancer, with the screening of prostate adenocarcinoma, in particular, presenting vulnerabilities in the detection methods utilized at the present time. It is estimated that approximately 1 in 7 men will develop prostate adenocarcinoma at some point in their lifetime thus making it a dominant health concern (1). Prostate cancer is the second leading cause of cancer death among men and remains the most common malignancy affecting males above the age of 40 in the United States (2,3). It is currently known that the incidence of prostate adenocarcinoma correlates with age, and as men get older, the importance of prostate cancer screening increases.

As it currently exists, the most prevalent screening methods for prostate cancer are blood tests for Prostate specific antigen (PSA) and Digital Rectal Exams (DRE) (4). PSA is a glycoprotein enzyme secreted by epithelial cells of the prostate to liquefy semen in the seminal coagulum (5). PSA is detected in small quantities in the serum of males with healthy prostate cells, but PSA levels are elevated in the presence of prostate cancer or other prostate disorders (6). Digital rectal exams are performed to locate bumps or hard areas on the prostate that are potentially cancerous, but the procedure is less effective than PSA blood testing and results in patient discomfort (3).

While elevated PSA in serum has been traditionally utilized as an indicator for prostate cancer, limitations of PSA blood tests are exhibited through data suggesting that serum PSA is also elevated for causes unrelated to prostate carcinoma such as prostatitis, benign prostatic hyperplasia, diet alterations, environmental conditions, and after minor clinical procedures such as transrectal ultrasound (7). In fact, the over-diagnosis of prostate cancer by as much 17-50% (8) has led the United States Preventative Services Task Force (USPSTF) to recommend against PSA-based detection for prostate cancer (9). Over-diagnosis of prostate cancer has often resulted in unnecessary, painful biopsies and surgical complications where only about 30% of patients were found to have high-grade carcinoma (7, 9,10).

The discovery of highly specific biomarker α-Methylacyl Coenzyme A Racemase (AMACR) and its consistent over-expression in the proliferation of malignant prostate cancer epithelium (10-13) has created an opportunity for new detection methods of prostate cancer to arise. AMACR, unlike PSA, has high sensitivity and specificity for prostate carcinoma, further distinguishing cancer cells from benign cells (10,11,13). Utilizing AMACR as a target in a sandwich Enzyme-linked Immunosorbent Assay (ELISA) may provide an avenue by which to develop a novel diagnostic for prostate cancer (see figure 1). An ELISA is a powerful method for detecting and quantifying the presence of a target molecule through the integration and binding of antibodies to amplify a biochemical signal (14). During an ELISA, antibodies are typically utilized to bind and immobilize the target, but their use is expensive and inefficient. Aptamers are oligonucleotides (RNA in this context) that may provide a better alternative to antibodies in ELISAs due to their affordability, ease of production, target specificity, and flexible secondary structures that result in high target affinity. Aptamers have a proven themselves in a variety of different applications such as drug delivery and diagnostics. Examples of applications include the aptamer Pegaptanib Sodium which targets vascular endothelial growth factor (VEGF)-165 for therapeutics against age-related macular degeneration (15), and a resonance sensor utilizes aptamers as components for the detection of subnanomolar thrombin (16). The discovery of aptamers against AMACR and Horseradish Peroxidase (HRP) would provide the components necessary to establish a new test for prostate cancer.

Horseradish Peroxidase, shown in figure 1, is often utilized in ELISAs for its ability to generate a colorimetric response (17) by catalyzing the oxidation of substrates through coupling with consumption of hydrogen peroxide (18). HRP itself is a 44.1739 kDa all alpha-helical enzyme derived from the roots of Armoracia rusticana, known by its common name as horseradish. Currently HRP is widely utilized in biochemical and medical applications as a reporter molecule to detect the presence of specific targets in a patient's blood.

At the current time, round three of aptamer selection is being performed to enrich oligonucleotides with high affinity and specificity towards HRP. Results from agarose gel electrophoresis indicate that the oligonucleotide concentration of W0 species is decreasing for each round, and the concentrations for W4 and E1 are increasing. These results signify progress in the potential discovery of an aptamer for HRP. A table of round selection conditions is shown in table 1. An aptamer for HRP would serve as a crucial element in the development of an ELISA for prostate cancer detection.

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References