Uma Vaidyanathan's GOx Aptamer Project

Aptamer Selection Against Glucose Oxidase: Building a More Efficient Glucometer

Introduction & Background

As of 2012, about 29.1 million Americans or 9.3% of the population struggle with diabetes (1). Additionally, the global prevalence of diabetes is progressively increasing, as it is projected that the number of people suffering from diabetes will increase from 171 million in 2000 to about 366 million in 2030 (2). Diabetes, characterized by sustained periods of high blood-glucose levels, remains as one of the most common diseases in the world. Patients suffering from diabetes use glucometers to measure their blood-glucose levels to manage their treatment and regulate their sugar intake. Current glucometers function by using test strips containing glucose oxidase (GOx), which test the presence of glucose in the blood. Glucose oxidase is an enzyme that catalyzes the oxidation of blood-glucose, specifically β-D-glucose, to gluconic acid and hydrogen peroxide (3). GOx is immobilized on the glucometer test strips, which are disposable after use. Glucose levels are measured indirectly through the production of a concentration of hydrogen peroxide. A sensor on the glucometers detects the hydrogen peroxide levels and prompts the numerical display of the relative amount of glucose in the blood sample (4). Current glucometers rely on disposable test strips and there is a possibility that the enzyme could malfunction due to its sensitivity. For example, a study by Eremin et al. in 2001 indicated that the GOx enzyme has a susceptibility to destabilize at lower temperatures and denature at higher temperatures. (5) A glucometer “off switch” or glucose oxidase inhibitor could alleviate some of this background signal. To address this, we seek to identify an aptamer inhibitor against GOx.

N71 selection against Glucose Oxidase.

Glucose oxidase is composed of two identical polypeptide chain subunits that are covalently bonded by disulfide bridges; each of the two polypeptide chains contain one mole of flavin-adenine dinucleotide and one mole of iron. Glucose oxidase (160kDa) is a dimer and has an active site for glucose, as it converts glucose to gluconic acid and hydrogen peroxide. The optimum pH for the enzyme is 5.5, though it is able to be efficient in solutions with a pH range of 4-7. The buffer for glucose oxidase is a 0.1M sodium acetate buffer with a pH of 4. The pI based on the amino acid sequence of glucose oxidase is 4.2 (6). Glucose oxidase has many applications as it is a powerful anti-microbial used in the food industry, can be used to track blood- glucose levels, and determine the amount of glucose in body fluids and foods (7). Recent research has shown the potential use of glucose oxidase in enzymatic kinetics when coupled to horseradish peroxidase (8). No aptamers have been selected for glucose oxidase yet.A more efficient glucometer can be created using an aptamer against the GOx enzyme. Aptamers, oligonucleotides capable of binding to specific molecules, are used in this project to inhibit its cognate analyte, GOx. An aptamer from the N71 pool will be selected that binds with a high affinity to the glucose oxidase protein.

Although antibodies have been used for similar functions, aptamers are easier to produce, especially as affinity reagents. In addition, aptamers are less expensive and, as a result, aptamer research can be more economical. These aptamers thus have a binding affinity for various molecular targets, such as proteins and other small molecules. For example, in 2008, a research project was successful in selecting aptamers against the mutant hiv-1 reverse transcriptase. In this study, aptamers were used in diagnostic and therapeutic advances (9).

When an aptamer binds to the protein, the oligonucleotide changes the conformation of the protein and thus acts as an inhibition mechanism. By using an oligonucleotide to inhibit the function of GOx in a glucometer pictured in Figure 3, we will be able to control when the glucometer is activated and thus preserve the function of the enzyme and remove background signal. Glucose oxidase, being an

enzyme that catalyzes the oxidation reaction converting glucose to gluconic acid and producing hydrogen peroxide in the process, can be considered one of the main enzymes responsible for the detection of glucose in the blood. When GOx is inhibited by the aptamer, GOx prevents the conversion of β-D-

glucose in the blood to gluconic acid and hydrogen peroxide. Thus, because hydrogen peroxide is not produced and not measured, there is no detection of glucose in the blood. This aptamer can serve as an inhibitor to preserve the function of GOx until it is needed. This ability to turn off the GOx enzyme will allow for the creation of a more efficient glucometer or possibly increase the shelf life of the glucometer

In vitro bead-based aptamer selection against GOx offers an effective solution to creating a more efficient glucometer or increasing the shelf life of the enzyme. The process, depicted in Figure 2, is performed using a bead-based method. Streptavidin

beads that have a high affinity for the biotinylated GOx are used to separate the target-bound RNA from the unbound RNA. The process involves immobilizing the protein target on the beads and to wash away the unbounded target. An RNA pool, which consists of various distinct RNA sequences, one of which could be a potential aptamer, is introduced to the immobilized target. The oligonucleotides that

bind to the target, or the bound RNA sequences, are eluted. DNA copies of the RNA pool are made using reverse transcriptase and cycle course PCR is used to determine the optimal number of cycles for the specific pool for amplification in large scale PCR. The resulting amplified dsDNA is transcribed to produce RNA. The RNA can then undergo purification using a PAGE gel and precipitation. Further

rounds of selection can be performed on the eluted RNA to identify the most efficient oligonucleotide in binding to the specific target of interest.

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Citations

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2) Wild, S., Roglic, G., Green, A., Sicree, R., & King, H. (2004). Global Prevalence of Diabetes: Estimates for the Year 2000 and Projections for 2030: Response to Rathman and Giani. Diabetes Care, 2569-2570.

3) Bankar, S., Bule, M., Singhal, R., & Ananthanarayan, L. (2009). Glucose oxidase — An overview. Biotechnology Advances, 27(4), 489-501.

4) Blood Glucose Meter Design | EE Times. (2010, July 13). Retrieved September 15, 2015, from http://www.eetimes.com/document.asp?doc_id=1278181

5) Eremin AN, Metelitsa DI, Shishko ZhF, Mikhaĭlova RV, Iasenko MI, Lobanok AG. [Thermal stability of Penicillium adametzii glucose oxidase]. Prikl BiokhimMikrobiol. 2001 Nov-

Dec;37(6):678-86. Russian. PubMed PMID: 11771321.

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7) Glucose Oxidase from Aspergillus niger. (n.d.). Retrieved September 15, 2015, from http://www.sigmaaldrich.com/catalog/product/sigma/g2133?lang=en®ion=US

8) Huang, Y., Chen, X., Xia, Y., Wu, S., Duan, N., Ma, X., & Wang, Z. (2013). Selection, identification and application of a DNA aptamer against Staphylococcus aureus enterotoxin A. Analytical Methods, (3), 690-697.

9) Bateman, R., & Evans, J. (1995). Using the Glucose Oxidase/Peroxidase System in Enzyme Kinetics. J. Chem. Educ. Journal of Chemical Education, 72(12). Retrieved September 15,2015.

10) Li, N., Wang, Y., Pothukuchy, A., Syrett, A., Husain, N., Gopalakrisha, S., . . . Ellington, A. (2008). Aptamers that recognize drug-resistant HIV-1 reverse transcriptase. Nucleic Acids Research, 36(21), 6739-6751. Retrieved September 15, 2015.

11) Kedzierski, S., Caltagirone, T., & Khoshnejad, M. (2013). Synthetic Antibodies: The Emerging Field of Aptamers. BioProcessing Journal BioProcess J, 46-49.

12) Glucose Oxidase. (n.d.). Retrieved September 15, 2015, from http://www.rcsb.org/pdb/101/motm.do?momID=77

13) Dijken, J., & Veenhuis, M. (1980). Cytochemical localization of glucose oxidase in peroxisomes of Aspergillus niger. European Journal of Applied Microbiology and Biotechnology European J. Appl. Microbiol. Biotechnol., 9(4), 275-283.

14) Yanez, M. (2013). Glucose Meter Fundamentals and Design - Freescale Semiconductor. Retrieved September 15, 2015, from http://cache.freescale.com/files/microcontrollers/doc/app_note/AN4364.pdf

15) Tonyushkina, K., & Nichols, J. (2009). Glucose Meters: A Review of Technical Challenges to Obtaining Accurate Results. Journal of Diabetes Science and Technology, 971-980.