Chris Harkness' GOx Aptamer Selection Project

Invitro, Filter-Based Aptamer Selection Against Glucose Oxidase for Diagnostic Test Strip for Pancreatic Cancer to Detect the Presence and Quantity of the Protein CA 19-9

Introduction/Background:

This year an estimated 56,770 people will be diagnosed with pancreatic cancer. It is one of the most difficult cancers to detect since the pancreas is located so deep in the body; therefore, making it extremely difficult to treat. Only 9% of people diagnosed with pancreatic cancer will survive 5 years after their diagnosis. It ranks in the top ten most common cancers in both men and women and is the seventh leading cause of cancer deaths (Rawla, Sunkara, & Gaduputi, 2019). Pancreatic cancer is a type of cancer located in the pancreas (“Why Is Pancreatic Cancer So Deadly?,” n.d.). Pancreatic Cancer is diagnosed by finding a tumor in the pancreas through different kinds of scans. For instance, an ultra-sound or CT scan. This means that by the time they are diagnosed it has had time to progress. A patient probably won’t even receive one of those scans until symptoms become prominent. One study led by Alexandra Newton of UC San Diego School of Medicine found that increased levels of the enzyme PHLPP1 lead to lower levels of Protein Kinase C (PKC) which is essential for many cell processes including survival. It was also found that patients with low levels of PKC survived for a much shorter amount of time linking high levels of PKC to a better chance at survival. The enzyme PHLPP1 works as a sort of “proofreader” and helps to regulate PKC levels. Currently research is being done to control PKC levels using the enzyme PHLPP1. This would give patients a better chance at survival. Finding an Aptamer to detect the presence of the cancer at an earlier stage would also allow for treatment to be administered more quickly and even possibly a chance at curing the disease (“Balance of Two Enzymes Linked to Pancreatic Cancer Survival,” n.d.).

An Aptamer is an Oligonucleotide (or piece of DNA/RNA) that best binds to a specific target protein. An easy way to picture this would be a lock and key, the Aptamer is selected against a target so that in a way it is specifically made for the target; Just like a is for a key. Aptamers have been shown to be very cheap and effective in many applications. Aptamers have been beneficial for different diseases through diagnostics, therapeutics and much more. Aptamer research has even been used in cancer research for a wide range of things. Since aptamers are so specialized, they are able to very effectively target different proteins related to different cancers. The target that will be used in this research is Glucose Oxidase or GOx. GOx is an enzyme that catalyzes the oxidation of glucose to hydrogen peroxide and D-glucono-1,5-lactone. This enzyme is used in glucometers, which are used to measure glucose concentrations in people with diabetes (Aptamer Target information page). GOx is can also be used as a reporter molecule. Therefore, it can be used to detect the presence of something else and indicate that it is there. Many reporter molecules turn florescent colors to indicate the presence of a specific biomarker or enzyme. GOx has a biotin affinity tag and a molecular weight of 160kDa. It is stored in the 4⁰C Fridge and obtained from Accurate Chemical and Scientific Corporation costing approximately $109 per 5mg. I hope to find an Aptamer to select against GOx to use in the diagnostic test strip for pancreatic cancer. This test strip would detect the presence of the cancer much earlier than it is able to be detected as of right now. Allowing for treatment to start earlier which would increase the chances of a successful treatment and most importantly survival. In order to find an aptamer DNA/RNA must be selected against a target. The method of selection that will be used in this research will be the SELEX method. To help visualize what the SELEX method does, a good example would be looking for gold. At first there is a large pool of DNA/RNA. This pool becomes less diverse through bead-based or filter-based selection. In this case invitro Filter-based selectin. In filter selection the RNA that finds to the target gets caught on the filter while the unbound RNA flows through the filter. Just like gold is caught in the pan and all the dirt, mud, and water flow through a filter. After a round of selection that bound RNA is amplified to make more copies and ran through another round of selection. To find an aptamer multiple rounds of selection must be completed and only the RNA that best binds to the target will be left. Aptamers selected against GOx have already been found in other research. One of which found that “Aptamer based ATP binding leads to the release of the cofactor FAD, which acts as a trigger to ‘turn-on’ the activity of apo-GOx and thus generate a measureable response”(Sitaula, Branch, & Ali, 2012).

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References

Balance of Two Enzymes Linked to Pancreatic Cancer Survival. (n.d.). Retrieved September 25, 2019, from UC Health—UC San Diego website: https://health.ucsd.edu/news/releases/Pages/2019-03-20-two-enzymes-linked-to-pancreatic-cancer-survival.aspx

Haglund, C., Kuusela, P., & Roberts, P. J. (1989). Tumour markers in pancreatic cancer. Annales Chirurgiae Et Gynaecologiae, 78(1), 41–53.

Hidalgo, M. (2010). Pancreatic Cancer. New England Journal of Medicine, 362(17), 1605–1617. https://doi.org/10.1056/NEJMra0901557

Kemppainen, E. A., Hedström, J. I., Puolakkainen, P. A., Sainio, V. S., Haapiainen, R. K., Perhoniemi, V., … Stenman, U.-H. (1997). Rapid Measurement of Urinary Trypsinogen-2 as a Screening Test for Acute Pancreatitis. New England Journal of Medicine, 336(25), 1788–1793. https://doi.org/10.1056/NEJM199706193362504

Li, C., Heidt, D. G., Dalerba, P., Burant, C. F., Zhang, L., Adsay, V., … Simeone, D. M. (2007). Identification of Pancreatic Cancer Stem Cells. Cancer Research, 67(3), 1030–1037. https://doi.org/10.1158/0008-5472.CAN-06-2030

Rawla, P., Sunkara, T., & Gaduputi, V. (2019). Epidemiology of Pancreatic Cancer: Global Trends, Etiology and Risk Factors. World Journal of Oncology, 10(1), 10–27. https://doi.org/10.14740/wjon1166

Sitaula, S., Branch, S. D., & Ali, M. F. (2012). GOx signaling triggered by aptamer-based ATP detection. Chemical Communications (Cambridge, England), 48(74), 9284–9286. https://doi.org/10.1039/c2cc34279k

Tumor Markers [CgvArticle]. (2019, June 26). Retrieved October 26, 2019, from National Cancer Institute website: https://www.cancer.gov/about-cancer/diagnosis-staging/diagnosis/tumor-markers-fact-sheet

Why Is Pancreatic Cancer So Deadly? (n.d.). Retrieved September 22, 2019, from https://healthcare.utah.edu/healthfeed/postings/2019/03/pancreatic-cancer.php

SELEX—a (r)evolutionary Method to Generate High-Affinity Nucleic Acid Glands. Stoltenburg, R., Reinemann, C., Strehiltz, B. https://d3i71xaburhd42.cloudfront.net/5898e293600547153311c0047c16784894e21b0f/3-Figure2-1.png