Abigail Weber's GOx Aptamer Selection Project (2018)

Aptamer for the Inhibition of Glucose Oxidase to Increase the Reliability of Glucometers

Introduction and Background

Diabetes is a disease that affects about 371 million people worldwide. Because of this, it is important that people have a reliable way to monitor blood glucose levels. Glucometers are a tool that diabetic patients to screen their blood glucose levels. They use test strips containing glucose oxidase, which converts glucose into materials that is easily detectable by machines. Glucometers are a great resource for diabetic patients, but the readings could be made more reliable by ensuring that nothing else is binding to the test strips, potentially contaminating the sample. In addition, the life of the test strip could be prolonged to ensure the strips will not expire. An inhibitory aptamer against glucose oxidase would prevent contamination and prolong the life of the test strips, bettering glucometers for patients around the world. Aptamers are oligonucleotide sequences that tightly bind to other molecules, such as proteins and enzymes. They have multiple purposes in diagnostics, therapeutics, and drug-delivery. In addition, they are cheaper and easier to product than antibodies. In this research project, an inhibitory aptamer against glucose oxidase will be isolated and used to function as a second-hand diagnostic to increase the sensitivity of glucometers by ensuring that nothing else is reacting with glucose oxidase, potentially effecting its function.

The inhibitory aptamer, when bound to glucose oxidase, would turn off the function of the enzyme. Glucose oxidase reacts with glucose to eventually make hydrogen peroxide, the molecule that is easily detectable by the platinum electrodes in glucometers. The amount of hydrogen peroxide in the blood sample correlates to the amount of glucose. Inhibiting glucose oxidase would guarantee that nothing else is binding to it while it is not being used. In addition, an inhibitory aptamer against glucose oxidase would increase the shelf life of the test strips. The aptamer would turn off the enzyme, making it non-functional and conserving its metabolic energy until it is ready to be used. When the test strips are ready to be used, the aptamer will immediately denature when the body temperature blood reacts with the room temperature test strip. Denaturation of the aptamer will cause the enzyme to be fully functional again, allowing glucose oxidase to make hydrogen peroxide.

The process of creating an aptamer will involve selection with the use of a filter. Glucose oxidase is a negatively charged molecule, making it harder for the enzyme to want to bind to a negatively charged nucleic acid. It is shown that it is more stable using a PBS buffer with MgCl₂ (5.7 mM Phosphate pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5 mM MgCl₂.) The filter partitions the target-bound RNA from the unbound RNA. Washes are performed in order to increase stringency and isolate the bound species. Reverse transcription is necessary in order for greater stability in amplification through polymerase chain reaction. Following transcription, the bound species is purified through polyacrylamide gel electrophoresis (PAGE). This purified bound species is then used for the next rounds of selection to enrich the pool used in selection. After many rounds of selection, bound species are able to undergo a binding assay to determine the presence of a strong binding affinity to the target.

Work up until spectroscopy has been accomplished. Previously, 3 PAGE and transcription reactions were completed, but transcription failed three times. Since there was no more large-scale PCR product to redo another transcription reaction, reverse transcription had to be repeated. Cycle course PCR, large scale, ethanol precipitation, and another transcription and PAGE were completed with success.

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References and Citations

Fast Facts - Data and Statistics About Diabetes. (n.d.). Retrieved March 29, 2018, from https://professional.diabetes.org/content/fast-facts-data-and-statistics-about-diabetes

Glucose Oxidase from Aspergillus nigerG7146. (n.d.). Retrieved March 30, 2018, from https://www.sigmaaldrich.com/catalog/product/sigma/g7146?lang=en®ion=US

Goodsell, D. (2006, May). Glucose Oxidase. Retrieved March 30, 2018, from http://pdb101.rcsb.org/motm/77

Statistics About Diabetes. (n.d.). Retrieved March 29, 2018, from http://www.diabetes.org/diabetes-basics/statistics/