Allen Ku's GOx Aptamer Selection Project (2018)

The Development of an RNA Aptamer Against Glucose Oxidase for a more Cost-Effective Diagnostic Tool for Diabetes

Introduction and Background

In recent years, aptamer technology and research have gained considerable grounds in the health and pharmaceutical industries. An aptamer is defined as an oligonucleotide that bind to specific target molecules as shown in Figure 1. Often times, aptamers and anti-bodies are so similar that they are comparable in terms of function. While both aptamers and antibodies do perform the same functions, there is a clear advantage in using aptamers over antibodies. Not only are aptamers relatively easy to make, they are also cheaper to develop and mass produce. This factor could significantly cut down the cost spent on producing anti-bodies while producing similar results. Another advantage of aptamers over anti-bodies is that aptamers are made in- vitro.

Other than laboratory applications, aptamers have made a huge impact on the pharmaceutical and health industry. Aptamers are commonly used as diagnostic tools to detect bacterial species or enzymes. Aptamer technology has the potential to even identify targets involved in various diseases, viral infections, and even cancer. The development of the Systematic Evolution of Ligands by Exponential enrichment (SELEX) allowed for the in vitro selection of aptamers against various targets of interest (Manley, 1970). The SELEX method is comprised of nine essential steps: target immobilization, selection, ethanol precipitation, reverse transcription, a cycle course polymerase chain reaction, (ccPCR) a large scale polymerase chain reaction (lsPCR), polyacrylamide gel electrophoresis, and quantification. The first round of selection identifies the most efficient RNA binders from an aliquot of N71 RNA pool and amplifies them. The RNA that yields from the first-round of selection then undergoes subsequent rounds of selection with increasing stringency to wash out the weaker RNA binders from each round. After multiple rounds of selection, the presence of an enriched sequence will indicate that an aptamer has been found. A binding assay will then be conducted to see how well the aptamer actually binds to the target of interest.

The purpose of this experiment is to select an aptamer against glucose oxidase from the N71 RNA pool. By making GOx the target enzyme, the concentration of glucose within the body can be monitored through the use of an enzyme linked oligonucleotide assay, or ELONA. The ELONA used in this project will utilize a capture antibody and an aptamer for glucose oxidase. A sample of blood will be introduced to a plate of anti-glucose antibody. The glucose in the blood will attach to the glucose antibody which will immobilize the glucose. The aptamer is then introduced and will bind to the immobilized glucose, producing a colorimetric effect. The concentration of glucose can then be monitored. Due to the inexpensive nature of producing aptamers, this method could potentially serve as a replacement for expensive glucometers and glucose test strips.

This selection is currently on its second round. Trouble-shooting tests conducted during the first round of selection used up most of the E1 solution. The selection was restarted to replenish the E1 solution. After starting over, round 1 of selection was completed and round 2 of selection subsequently began and is now on the transcription step. Once transcription is completed, the product will be subjected to a polyacrylamide gel electrophoresis to isolate and purify the RNA. Once the RNA is quantified, more rounds of selection will follow, with the goal of developing a GOx aptamer that will serve as a more cost-effective diagnostic tool for diabetes.

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Works Cited

American Diabetes Association. (2013). Economic Costs of Diabetes in the U.S. in 2012. Diabetes Care, 36(4), 1033–1046. http://doi.org/10.2337/dc12-2625

Centers for Disease Control and Prevention. National Diabetes Statistics Report, 2017. Atlanta, GA: Centers for Disease Control and Prevention, U.S. Dept of Health and Human Services; 2017.

Liu, X. M., Federlin, K. F., Bretzel, R. G., Hering, B. J., & Brendel, M. D. (1991, July). Persistent reversal of diabetes by transplantation of fetal pig proislets into nude mice. Retrieved July 14, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/2060721

Manley, J. L. (1970, January 01). SELEX to Identify Protein-Binding Sites on RNA. Retrieved July 14, 2018, from http://cshprotocols.cshlp.org/content/2013/2/pdb.prot072934.full

PDB101: Molecule of the Month: Glucose Oxidase. (2006, May). Retrieved July 15, 2018, from http://pdb101.rcsb.org/motm/77

What is Diabetes? (n.d.). Retrieved July 14, 2018, from https://www.diabetesresearch.org/what-is- diabetes?gclid=Cj0KCQjwvqbaBRCOARIsAD9s1XBzPqv9Q6eyl5rqztWrM_PWhLocq9IcS DkRQpjaxPSX_0IDo6JloaoaAo_8EALw_wcB