Sandhya Srinivasa's GOx Aptamer Project (2017)

RNA Aptamer Selection Against Glucose Oxidase to Increase the Efficiency of Biosensors

Introduction and Background

Diabetes is a prevalent issue, particularly in the United States and involves two different types, Type 1 and Type II. To diagnose diabetes, glucometers consisting of the enzyme glucose oxidase found in test strips, which are used to measure blood glucose levels. Glucose oxidase is an enzyme that converts oxygen into hydrogen peroxide while converting glucose into glucolactone (Goodsell, 2006). The technology present in glucometers takes advantage of this side reaction that occurs in which oxygen is converted into hydrogen peroxide in order to measure the blood glucose concentration to diagnose diabetes (Goodsell, 2006).

Despite this current technology, present diabetes diagnostic approaches are not as accurate in determining blood glucose levels. This issue can be solved by increasing glucose oxidase’s affinity for glucose; this would add to more specificity in the binding at the active site and would produce an accurate quantity of hydrogen peroxide. This reaction that occurs at the active site of glucose oxidase involves the oxidation of glucose into glucolactone with the aid of the FAD cofactors, indicated by the red regions in Figure 2 (Goodsell, 2006). As can be seen from Figure 2, the glucose oxidase target is a dimer made up of two identical subunits containing FAD and it is this FAD cofactor that that is reduced to FADH2 (McDowall, 2006). The FADH2 is then oxidized to allow for the reduction of oxygen into hydrogen peroxide (McDowall, 2006).

Clearly, the FAD cofactor shown in Figure 2 is an integral part of the target protein. Because of its importance, glucose oxidase is an enzyme found not only in humans, but also in fungi due to its ability to oxidize glucose while producing hydrogen peroxide, providing antibacterial properties (Goodsell, 2006). In this way, the hydrogen peroxide provides a protection for the fungi against bacteria (Goodsell, 2006). Since glucose oxidase converts glucose while also producing hydrogen peroxide, it has become extremely important in measuring blood glucose concentrations using glucometers (Goodsell, 2006). With these glucometers, the use of glucose oxidase and its FAD cofactor makes it simple to measure the amount of hydrogen peroxide to then determine the amount of glucose in the blood (Goodsell, 2006). Due to the importance of glucose oxidase, several labs have produced glucose oxidase recombinantly in the fungi Aspergillus niger and Penicillium (Ferri, 2011).

Because of the importance of glucose oxidase in glucometers and in fungi, it is necessary to find an aptamer for this target. Currently, there is no aptamer found for glucose oxidase for a specific diagnostic function. An aptamer is essentially an oligonucleotide, either an RNA or DNA strand, that binds onto the target protein and has a high affinity for it, generally in order to inhibit the protein. Due to its high affinity for a target protein, it binds more effectively than an antibody and is a cost effective alternative.

Finding an aptamer would prove to be beneficial in increasing the accuracy of current glucometers in diagnosing diabetes and discovering novel diabetes diagnostic tools. It may even be possible to use glucometers to measure the concentration of glucose using glucose oxidase and to diagnose diabetes within urine itself if there is sufficient concentration of detectable hydrogen peroxide; this would be possible if there is a high affinity between glucose and glucose oxidase. Current glucometers can also be improved upon as glucose oxidase can also oxidize other monosaccharides within blood. This means that current diagnostic techniques in glucometer readings are not as accurate and may include other monosaccharides such as xylose and gluconolactone in blood (Wilson, 1991). Furthermore, other experimenters found issues with preventing maltose and isomaltose from binding to glucose oxidase instead of allowing glucose to bind to the enzyme and were unsuccessful in remedying this problem (Dahlqvist, 1961). To solve this issue, an aptamer can be found that binds to glucose oxidase to increase the specificity of the enzyme’s active site for glucose oxidase to provide a more accurate reading.

The SELEX process shown in Figure 3 is a key process that can be used to find this aptamer. The SELEX process consists of numerous steps involving target immobilization, binding and selection, RNA elution, ethanol precipitation, reverse transcription, cycle course PCR (ccPCR) and gel, large scale PCR (lsPCR) and gel, ethanol precipitation, transcription, and finally polyacrylamide gel electrophoresis (PAGE). Target immobilization involves immobilizing glucose oxidase with Streptavidin magnetic beads which are biotinylated. Then, binding and selection involves performing three washes using the HEPES buffer in this case to wash away any unbound RNA from the pool that did not bind to glucose oxidase and the RNA was eluted. Since glucose oxidase has an optimum pH ranging from 7-7.5, the selection buffer of HEPES with a pH of 7.3 was chosen. Ethanol precipitation is then performed to concentrate the eluted RNA and reverse transcription was performed to convert the RNA to ssDNA. Cycle course PCR involves producing dsDNA from the ssDNA to determine the number of cycles the template amplifies at without overamplifying from the ccPCR gel. Then, lsPCR is performed at the specific cycle from ccPCR and transcription was performed to convert the dsDNA into RNA. Finally, PAGE is performed to visualize the RNA and to purify the RNA. Then, the RNA concentration could be measured with the nanodrop spectrophotometer. This same SELEX procedure could then be repeated around 7 other times to find an aptamer for glucose oxidase.

Once this aptamer is found, DNA microparticles can be synthesized which consist of ssDNA with aptamer binding sites to deliver the aptamer to glucose oxidase in the glucometer test strips. These ssDNA can be cross linked to form a sphere, producing DNA microparticles that consist of aptamer binding sites that are complementary to the aptamer sequence (Kim, 2016). This DNA microparticle can effectively deliver the aptamer to the target protein glucose oxidase found in the test strips of the glucometer. Some studies using aptamers have shown that some aptamers can increase the specificity as can be seen with Kim’s study involving thrombin, which helped increase its efficiency around 1.7 times (Kim, 2016). Because of the success of using DNA microparticles in increasing the specificity of thrombin, it can be deduced that DNA microparticles may also be used to increase the specificity of glucose oxidase present in test strips of glucometers to increase the overall accuracy of the glucometer. These DNA microparticles are extremely beneficial and efficient as it contains several aptamer binding sites and can bind to several glucose oxidase enzymes to increase its specificity in the test strips (Kim, 2016). Therefore, only a small amount of DNA microparticles need to be constructed to increase the accuracy of the glucometer in only detecting blood glucose.

Once this DNA microparticle-aptamer complex is produced, it can then be introduced to glucose oxidase found in the test strips of the glucometer as the patient can take a pill consisting of this aptamer-DNA microparticle complex. By doing so, the aptamer-DNA microparticle complex could potentially circulate in the blood stream and when measuring blood glucose levels through test strips, the blood containing this aptamer-DNA microparticle complex would be able to act on the glucose oxidase found on the test strips. This complex could potentially bind to glucose oxidase as it consists of mostly B pleated sheets that are found on the exterior and at the surface of the protein, establishing polarity and making the enzyme amphiphilic (Schomburg, 1993). Because of this polarity, the complex could potentially bind to the portions of the B pleated sheets. Glucose oxidase also consists of the amino acids lysine and arginine, which are both positively charged, though it is mostly dominated by amino acids that take on a negative charge (Schomburg, 1993). However, it could still be possible for the negatively charged aptamer found in the complex to bind to the positively charged amino acids that are present within glucose oxidase to increase its specificity. Once the specificity of glucose oxidase increases, it will be able to accurately predict blood glucose levels via glucometers.

As of now, first round of aptamer selection using the mock pool for CIAP was completed and the aptamer selection for glucose oxidase is currently underway and continued from the previous semester. However, due to human error and loss of the products from the aptamer selection from the previous semester, the first round of aptamer selection is conducted once again from the beginning and, as of now, cycle course PCR is being conducted. Currently, the process of finding an RNA aptamer for glucose oxidase is still ongoing and in the future a total of six to eight rounds of selection will be completed to find an aptamer. Then, a binding assay will be performed and the aptamer will be sequenced. Using this aptamer sequence, a DNA microparticle will be produced complementary to the aptamer sequence and the DNA microparticle-aptamer complex will be produced. Then, this complex will be introduced to glucose oxidase in order to increase its specificity. As stated earlier, the research objective is to find an aptamer that binds to the glucose oxidase to increase its affinity at its active site with glucose to allow for more accurate glucometer readings and glucose concentration readings to effectively diagnose diabetes.

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References

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Dahlqvist, A. “Determination of Maltase and Isomaltase Activities with a Glucose-Oxidase Reagent” 80 (February 27, 1961): 547–51.

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Protein Data Bank. “Glucose Oxidase and biosensors.” May, 2006. Accessed April 5, 2017. https://www.ebi.ac.uk/interpro/potm/2006_5/Page1.htm.

Schomburg, D. “The 3D Structure of Glucose Oxidase from Aspergillus Niger. Implications for the Use of GOD as a Biosensor Enzyme” 8, no. 3–4 (1993): 197–203.

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Worthington Biochemical Corporation. “Glucose Oxidase.” Accessed April 5, 2017. http://www.worthington-biochem.com/gop/.