Joanna Lee's GOx Aptamer Selection Project (2019)

RNA Aptamer Against Glucose Oxidase for the Treatment of Hyperglycemia in Severe Diabetics Through Glucose Oxidation

Introduction and Background

Type 2 diabetes mellitus (T2DM) is the most common form of diabetes mellitus globally, accounting for 90-95% of diabetic cases, and its effects will continue to increase in the next twenty years (Wu, 2014). If not treated in time, this disease causes chronic hyperglycemia, which can lead to retinopathy, nephropathy, and cardiovascular diseases caused by oxidative stress when non-enzymatic glycosylation reactions are more easily induced by excessive blood glucose (Wu, 2014; Kawahito, 2009). Once a patient develops these downstream conditions, it becomes exceedingly difficult to treat due to the prices involved in treating these problems. Although there is medication used to prevent downstream effects of chronic hyperglycemia, patients may not be able to afford prescribed medication, such as insulin or metformin, due to the price inflation of medication and medical services (Peter, 2016). There may be, however, cheaper alternatives to these prescribed medications.

To address these extreme acute cases of elevated blood sugar levels, it may be possible to remove excess glucose in blood by using glucose oxidase. Glucose oxidase (GOx) is an enzyme, found in Aspergillus niger but not in humans, that oxidizes glucose into D-glucono-delta-lactone and hydrogen peroxide (Ferri, 2011). Glucose oxidation uses flavin adenine dinucleotide (FAD) as a cofactor to form gluconolactone, which can be a potential binding spot for other nucleic acids (Ferri, 2011) (refer to Figure 1).

Figure 1: Shows the process of glucose oxidation by glucose oxidase into D-glucono-delta-lactone and hydrogen peroxide (Szweda n.d.)

Glucose oxidase could be used to decrease excessive blood glucose levels. To do this, however, there needs to be a way to inhibit the oxidation reaction after a certain concentration of hydrogen peroxide is reached, due to the cytotoxic nature of hydrogen peroxide in humans (Halliwell, 2000). Knowing this, there is a possibility of using aptamers to inhibit the oxidation reaction. Aptamers are binding oligonucleotides that bind with high affinity to targets (McKeague, 2012). Finding an aptamer that binds to the FAD binding site on glucose oxidase will prevent the enzyme from oxidizing more glucose, inhibiting the reaction. Since the blood pH of those with diabetes mellitus has a lowered pH due to ketoacidosis (<7.35), an aptamer complex could be set up so oxidation can continue until blood pH is at regular physiological conditions (ranging from 7.35-7.45) (Marunaka, 2015). Still, a problem remains in that there will be residual hydrogen peroxide in human blood after the oxidation reaction is inhibited. To account for this, another aptamer bound to a hydrogen peroxide depleting target (such as the DNA aptamer for hemin, a peroxidase (Travascio, 1998)) could be found to create an aptamer-aptamer bounded complex to remove hydrogen peroxide from blood (Halliwell, 2000). This will decrease the blood glucose levels in extreme diabetics to decrease the chances of glycosylation reactions and prevent hydrogen peroxide poisoning.

The aptamer complex will consist of two aptamers (one for GOx and one for the hydrogen peroxide depleting protein), which can bind to each other through hydrogen bonding. This complex will begin with the GOx aptamer bound to the H2O2 depleting protein aptamer that is already bound on its target to account for the quick degradation and renal filtration time of aptamers in blood (Lakhin, 2013). The GOx aptamer should then be activated to bind to GOx to inhibit glucose oxidation after normal physiological pH is reached (pH 7.35-7.45) (Marunaka, 2015). This activation will cause the complex to bind to GOx to inhibit the reaction. The activity of the H2O2 depleting protein should be active even throughout the oxidation reaction to eliminate the possibility of hydrogen peroxide poisoning (refer to Figure 2).

Figure 2: Shows the process of the aptamer complex where: 1. Glucose will bind to GOx; 2. Glucose will break down into hydrogen peroxide and d-glucose-delta-lactone; 3. Hydrogen peroxide will be broken down by the secondary target into water and oxygen; 4. Previously lowered blood pH from ketoacidosis (<7.35) is raised above 7.35 (Marunaka, 2015); 5. GOx aptamer is activated to bind to GOx to inhibit glucose oxidation; 6. Any excess hydrogen peroxide in blood will be broken down into water and oxygen.

Using aptamers for this application allows for specificity in binding to glucose oxidase along with more molecular stability (Lakhin, 2013). To select for specifically binding oligonucleotides, the systematic evolution of ligands by exponential enrichment (SELEX) method was used. This method involves the binding of targets to high affinity binding oligonucleotides over several ‘rounds’ to isolate increasingly enriched pools of specifically target-binding RNA (Manley, 2013). After selecting for bound species, the sample is reverse transcribed into ssDNA, amplified through large scale PCR, transcribed back into RNA, and purified through polyacrylamide gel electrophoresis to generate enriched RNA pools of correct folding and begin new ‘rounds of selection’.

Currently, the second round of selection is in process, using a diluted RNA pool from the first round of selection. This second round will use filter-based selection to wash out bead-binding species not bound to glucose oxidase. The first round of aptamer selection using streptavidin beads has been completed, ending with a final concentration of RNA in the reformed RNA pool of 86.45 uM. As more rounds are done, each isolated RNA pool can be used for further rounds of selection to eventually isolate an aptamer from the RNA pool.

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References

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