Paul Nguyen's GOx Aptamer Product

Enhancing Glucometer Function: Aptamer against Glucose Oxidase

Introduction & Background

Diabetes is currently one of the most common diseases in the United States. From 1990 to 2008, the incidence and prevalence of diabetes had doubled; increases in diabetes have paralleled increases in obesity (Geiss LS et al., 2014). Diabetes is associated with conditions such as hypoglycemia, hypertension, stroke, and dyslipidemia. Due to this, people with diabetes rely on accurate glucometers in order to quickly assess their current blood sugar levels and respond appropriately. If a glucometer provides an inaccurate reading, or becomes completely nonfunctional, then the health of an individual with diabetes is put at risk. Because of this, it is necessary to find methods of improving the stability of the compounds that facilitate the glucometer’s mechanism of action (specifically, glucose oxidase).

Glucose oxidase (often abbreviated GOx, GO, or GOD) is an enzyme commonly harvested from the fungus Aspergillus niger. GOx (shown in Figure 1) is a dimeric protein comprised of two polypeptide subunits, with each subunit possessing a molecular weight of approximately 80 kDa. In addition, GOx is also one of the three main enzymes used to provide glucometers with their ability to measure glucose levels (Tonyushkina and Nichols, 2009). By catalyzing the oxidation of β-D-glucose to hydrogen peroxide, GOx provides a means for glucose levels in blood to be measured indirectly through hydrogen peroxide concentrations. Labs such as the Food Research Laboratory in Finland are researching GOx for unrelated reasons; besides applications in glucose level measurement, glucose oxidase also has antibiotic activity due to hydrogen peroxide production (Tiina and Sandholm, 1989) and can also improve the stability of bread dough (Decamps et al., 2012). Unfortunately, as with all enzymes, glucose oxidase is susceptible to denaturing under conditions of extreme temperatures. If glucose oxidase were stabilized, the performance of a glucometer would improve, even when exposed to extreme conditions.

Aptamers are short DNA or RNA sequences that have high binding affinities for specific protein targets (Ellington and Szostak, 1990). These molecules are beneficial due to their high specificity and versatility (Stoltenburg et al., 2007). Aptamers have applications in drug delivery, diagnostics, and therapeutics. By binding to targets, aptamers can inhibit dangerous molecules or enhance the effectiveness of a drug that is intended to be localized to the target (Xiang et al., 2015). An RNA aptamer with a high binding affinity for glucose oxidase is possibly capable of stabilizing the protein structure, allowing for greater resistance to unfavorable environmental conditions. The aptamer could then consequently be removed when the glucometer would need to be activated, possibly by using RNase to degrade the aptamer. This would reduce the likelihood of a faulty glucometer putting a person with diabetes in danger due to lack of accurate blood sugar information.

By using a bead-based in vitro selection method (depicted in Figure 2), an aptamer can potentially be discovered. An RNA pool containing a wide variety of sequences may contain a possible aptamer. The RNA sequences that bind to the target of interest are amplified via reverse transcription (which generates DNA) and PCR (which amplifies DNA). The resulting DNA can then be transcribed to generate RNA. After purification, the RNA can then undergo further rounds of selection. Though this target does not have any specific features that facilitate the binding of RNA besides the glucose binding active site, it may still be possible for an aptamer to be found. No aptamers have been found for glucose oxidase yet, which means that finding an aptamer for glucose oxidase will be a novel development. Ultimately, the objective of this research is to select for a stabilizing aptamer that will preserve the function of glucose oxidase, enabling glucometers to maintain accurate functionality when used to measure blood glucose levels.

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Citations

Decamps, K., Joye, I.J., Courtin, C.M., and Delcour, J.A. (2012). Glucose and pyranose oxidase improve bread dough stability. J. Cereal Sci. 55, 380–384.

Ellington, A.D., and Szostak, J.W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822.

Geiss LS, Wang J, Cheng YJ, and et al (2014). Prevalence and incidence trends for diagnosed diabetes among adults aged 20 to 79 years, United States, 1980-2012. JAMA 312, 1218–1226.

Stoltenburg, R., Reinemann, C., and Strehlitz, B. (2007). SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol. Eng. 24, 381–403.

Tiina, M., and Sandholm, M. (1989). Antibacterial effect of the glucose oxidase-glucose system on food-poisoning organisms. Int. J. Food Microbiol. 8, 165–174.

Tonyushkina, K., and Nichols, J.H. (2009). Glucose Meters: A Review of Technical Challenges to Obtaining Accurate Results. J. Diabetes Sci. Technol. Online 3, 971–980.

Wohlfahrt, G., Witt, S., Hendle, J., Schomburg, D., Kalisz, H.M., and Hecht, H.J. (1999). 1.8 and 1.9 A resolution structures of the Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes. Acta Crystallogr. D Biol. Crystallogr. 55, 969–977.

Xiang, D., Shigdar, S., Qiao, G., Wang, T., Kouzani, A.Z., Zhou, S.-F., Kong, L., Li, Y., Pu, C., and Duan, W. (2015). Nucleic acid aptamer-guided cancer therapeutics and diagnostics: the next generation of cancer medicine. Theranostics 5, 23–42.