Klayme-Shane Boucher's GOx Aptamer Selection

Aptamer Selection Against Glucose Oxidase for Glucometer Modifications

Introduction and Background

According to the American Diabetes Association, 29.1 million Americans in 2012, approximately 9% of the total population, had diabetes (American Diabetes Association, 2014). In the same year, it was projected the total cost of diagnosed diabetes to be 245 billion, 12% resulting from diabetes supplies. (Petersen, 2013). Among those supplies are glucometer expenses, including the purchasing of disposable test strips. Moreover, the accuracy of these test strips, which contain immobilized glucose oxidase, is still a clinical concern today. Thus, it is imperative that modifications to current glucometers are made to not only increase their efficiency, but to make them more economical as well. The development of aptamers, short strands of DNA that have a high binding affinity to a specific molecular target, will provide a solution to this problem (Stoltenhurg, Reinemann, & Strehlitz, 2007).

Glucose Oxidase, the molecular target in question here, is a dimer found in the fungus Aspergillus niger. Weighing at 160K Daltons, each glucose oxidase molecule consists of two polypeptide chains containing one mole of iron and one mole of flavin-adenine dinucleotide (FAD) (Raba, Horacio, & Mottola, 1995). The primary function of this enzyme is to oxidize glucose, more specifically beta-D glucose, into gluconic acid and hydrogen peroxide . Current glucometers use glucose oxidase as a means to produce hydrogen peroxide, which can then be detected and react with a dye. The color change resulting from the reaction is proportional to the concentration of glucose in the blood solution and thus is a method of measuring glucose levels (Tonyushkina & Nichols 2009).

Aptamers, as mentioned earlier, are single-stranded DNA or RNA molecules that bind to targets with high specificity and affinity. Discovered only a few decades ago, aptamers are an ermging class of oligonucleotides with the potential to serve as therapeutic and diagnostic agents. They also have proven to be successful in the past. In 2004, Macugen, the first aptamer-based drug, was discovered and is currently used to treat age-related Macular Degeneration (AMD), an eye disease that causes vision loss. The aptamer here inhibits interactions between Vascular Endothelial Growth Factors (VEGF) and their receptors (Lee, Jucker, & Pardi, 2008). Similarly, in this proposed application, an aptamer against glucose oxidase will inhibit the enzyme. Nevertheless, despite a comparable function, its application shall be completely different.

The aptamer against glucose oxidase would not change the enzyme’s function, but instead preserve it. In the proposed glucometer modification, the aptamer would inhibit glucose oxidase and therefore allow it to reside within the glucometer itself instead of within disposable test strips. Once the glucometer is turned on, a mechanism, such as a voltage or a change in pH, would separate the aptamer from the glucose oxidase and let the enzyme resume its function in oxidizing the glucose in the blood. Thus, the aptamer serves as a means of reducing the expense of glucometers by allowing them to use a single, permanent test strip.

Besides a glucometer modification, an aptamer against glucose oxidase could also potentially be used in an indirect enzyme linked immunosorbent assay (ELISA). ELISA is a diagnostic technique, which typically has used antibodies to detect certain molecules such as surface antigens (Toh, Citartan, Gopinath, & Tang 2014). In this process, however, aptamers would replace the antibodies in favor of their ability to be synthesized in vitro, their easy modifications, and their more economical production (Toh, Citartan, Gopinath, & Tang 2014). In this ELISA, an aptamer for glucose oxidase would bind complementary with another aptamer for a particular antigen. Glucose oxidase would serve as a reporter molecule by inducing a color change once the other aptamer has bound to the antigen. This assay, in the end, would be more favorable than attempting to produce one aptamer with high affinity for both targets (Toh, Citartan, Gopinath, & Tang 2014).

Finding an aptamer for glucose oxidase could prove to be problematic. The enzyme has no special features that favor the binding of nucleic acids (Raba, Horacio, & Mottola, 1995). In fact, it is very negatively charged and therefore discourages the binding of RNA or DNA. Moreover, in order for the enzyme to be stable, it would need to be stored in an acidic buffer. For the application at hand, such buffer could not be used, as the aptamer needs be functional at the human body pH. An alternative buffer will therefore need to be used.

Despite the potential problems, a glucose oxidase aptamer is underway. As of this moment, six rounds of aptamer selection have been conducted successfully. Ideally, aptamers for particular targets are found between 6-20 rounds of selection. Thus, the amount of rounds left for glucose oxidase is uncertain. Nevertheless, selection will continue until potential aptamer candidates are sequenced and utilized in a binding assay.If binding assay results are promising, the proposed glucometer modification may finally become a reality.

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Citations

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