Monika A. Rodriguez's GOx Aptamer Selection Project (2018)

The Development of an RNA Aptamer Against Glucose Oxidase: An Alternative Cancer Therapy

Introduction and Background

About 1,735,350 cases of cancer are diagnosed yearly – 609,640 cases resulting in death (National Cancer Institute, 2018). Current treatment options include surgery, hormone therapy, immunotherapy, and chemotherapy. Despite success, these treatment options can be costly and affect healthy cells along with cancerous ones – taking a heavy toll on the body. Some side effects of chemotherapy – which is one of the most commonly used therapies – include nausea, hair loss, and tiredness (Love, Leventahl, Easterling, Nerenz, 1989). Cancer cells fuel their own growth by consuming amino acids, glutamine, and glucose, but glucose can account for up to 15% of carbon found in those cells (Trafton, 2016). Because glucose’s contribution to the growth of cancer cells is so significant, it makes it the best food source to starve cancerous cells of. Being able to selectively induce apoptosis in the targeted cancer cells could greatly mitigate the side effects of current cancer therapies. An aptamer against glucose oxidase will be sought after in an effort to find a less expensive and less damaging alternative to the current selection of cancer treatment options for patients.

An aptamer is an oligonucleotide molecule selected from an RNA pool of random sequences that can bind to a selected target. Similar to an antigen binding to an antibody, an aptamer can be selected to bind an antigen without the biological system typically necessary for an antibody and for a fraction of the price. Monoclonal antibody drugs are currently used as a cancer therapy and are meant to imitate the immune system’s defense against cancer (Mayo Clinic, 2018). An aptamer against GOx could take the place of the antibodies (Figure 1) and bind to the antigens on cancerous cells to signal the attraction of GOx, which would ideally oxidize the glucose feeding the cancer and destroy it instead with the production of hydrogen peroxide with minimal risk of an immune response.

Glucose Oxidase is an enzyme produced in certain species of fungi, such as Aspergillus niger, and insects. As shown in Figure 2, It catalyzes the oxidation of glucose to hydrogen peroxide, thus enabling its anti-bacterial and anti-fungal properties (Wong, Wong, and Chen, 2008). GOx is a dimeric protein with a molecular weight of ~160 kDa. It is currently used as a biosensor in glucometers to measure glucose levels in diabetics. Among many others, a lab researching GOx is the Food Engineering and Technology Department at the University of Mumbai, where GOx is being produced by fermentation and recombination (Bankar et al., 2009).

Although a very negatively charged protein, GOx is proven stable within the pH range of 4-7. Its deep pockets could serve as a binding site for an aptamer. Its storage buffer is NaoAc because it becomes negatively charged with any higher of a pH, and its selection buffer was PBS – where it withholds a pH of about 7.4 and proves to be potentially stable in blood. No aptamer has been developed for Glucose Oxidase, and definitely not within this proposed application, but it has served in the development of an aptamer-thrombin complex (Golub et al., 2011).

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References

Bankar, S., Bule, M., Singhal, R. and Ananthanarayan, L. (2009). Glucose oxidase — An overview. Biotechnology Advances, 27(4), pp.489-501.

Golub, E., Freeman, R., Niazov, A. and Willner, I. (2011). Hemin/G-quadruplexes as DNAzymes for the fluorescent detection of DNA, aptamer–thrombin complexes, and probing the activity of glucose oxidase. The Analyst, 136(21), p.4397.

Love, R., Leventhal, H., Easterling, D. and Nerenz, D. (1989). Side effects and emotional distress during cancer chemotherapy. [online] Wiley Online Library. Available at: https://onlinelibrary.wiley.com/doi/abs/10.1002/1097-0142(19890201)63:3%3C604::AID-CNCR2820630334%3E3.0.CO;2-2.

Mayo Clinic. (2018). Monoclonal antibody drugs for cancer: How they work. [online] Available at: https://www.mayoclinic.org/diseases-conditions/cancer/in-depth/monoclonal-antibody/art-20047808.

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Polakis, P. (2015). Antibody Drug Conjugates for Cancer Therapy. Pharmacological Reviews, 68(1), pp.3-19.

Sigma-Aldrich. (2018). Glucose Oxidase from <I>Aspergillus niger</I> G6125. [online] Available at: https://www.sigmaaldrich.com/catalog/product/sigma/g6125?lang=en&region=US

Trafton, A. (2016). How cancer cells fuel their growth. [online] MIT News. Available at: http://news.mit.edu/2016/how-cancer-cells-fuel-their-growth-0307

Tuerk, C. and Gold, L. (1990). Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science, [online] 249(4968), pp.505-510. Available at: http://science.sciencemag.org/content/249/4968/505.long.

Wong, C., Wong, K. and Chen, X. (2008). Glucose oxidase: natural occurrence, function, properties and industrial applications. Applied Microbiology and Biotechnology, 78(6), pp.927-938.