Jaclyn Tran's GOx Aptamer Project (2016)

Diabetes is a term to talk about a group of metabolic diseases that result in hyperglycemia, or an excess amount of sugar in the blood. There are various different kinds of diabetes but Type 2 diabetes accounts for 90 to 95% of diabetes cases in the United States. Type 2 diabetes is also known as insulin resistance diabetes because the body is not sure how to process insulin. Insulin is used to return blood glucose levels back to normal by allowing glucose to enter cells in the body, so when there is insulin resistance the pancreas attempts to overcome this by creating an excess amount of insulin (American Diabetes Association, 2015). This excess of insulin builds up in the blood stream along with proinsulin, or islet amyloid polypeptides (IAPP/Amylin) (Pdb101, 2006).

Amylin (IAPP) is a peptide hormone that is secreted simultaneously along insulin by pancreatic cells, in order to regulate blood sugar spikes by gastric emptying. Amylin is one of the regulatory hormones that are required for cells to uptake glucose from the bloodstream (Scherbaum, 1998). Proislet amyloid polypeptide (proIAPP/proislet protein) is a protein that is also produced in the pancreas and helps to produce amylin through post-transitional modifications (Marzban et al., 2008), as shown in figure three. By utilizing an aptamer in an enzyme linked oligonucleotide assay that binds to glucose oxidase, in theory, physicians will be able to diagnose early onset diabetes type 2 earlier than they normally would with a traditional glucometer.

Glucose oxidase is an enzyme that uses FAD to transfer electrons that catalyzes glucose to D-glucono- δ- lactone and releases hydrogen peroxide as a byproduct of the reaction (see Figure 4) (Wong et al., 2008). It is produced by fungi such as Aspergillus niger, as well as various forms of bacteria (Pazur et al., 1964). It is a homodimer with a molecular weight of roughly 160 kDa, with an extremely negative charge at physiological pH. Glucose oxidase is not an enzyme typically found in the human body, because hydrogen peroxide is not present in the body. Glucose oxidase has a variety of useful applications for humans such as their presence in glucometers, additives to food to extend shelf life as a preservative, and for biosensors and biological assays (Ebi, 2016). Current research of glucose oxidase includes using GOx as a reporter molecule by measuring the amount of hydrogen peroxide released as a byproduct or pairing it with a colorimetric response to report the presence of glucose of urine (Radhakumary, 2011).

An enzyme-linked immunosorbant assay (ELISA) is used to determine if a specific protein is present. A secondary ELISA test determines how much protein is bound by a particular antibody (see Figure 5) (Davidson, 2002). By replacing the antibodies in an ELISA with aptamers, an enzyme-linked oligonucleotide assay (ELONA) is formed. A secondary ELONA would work with with an aptamer that will bind to glucose oxidase, functioning as a report molecule, and the proIAPP aptamer will bind to proIAPP proteins. As the amount of proIAPP in the blood increases, they will begin to bind to the proIAPP aptamer which is bound to the glucose oxidase aptamer. The glucose oxidase will function as a reporter molecule, releasing a colorimetric response that can be measured with a spectrophotometer to determine when there is an increase in proIAPP, therefore in insulin, within the bloodstream.

An aptamer is an oligonucleotide sequence that has a high binding affinity to a specific protein target with variety of uses. Aptamers have the possibility to replace the use of antibodies in binding assay since they are cheaper and safer for the body than triggering an immune response to receive antibodies. They can be used as therapeutic devices or as diagnostic devices. This experiment is focused on obtaining an aptamer to use in an ELONA test to bind glucose oxidase to proislet amyloid polypeptide in order to recognize a buildup of proIAPP in the blood. Helpful features of glucose oxidase include nucleotide binding site that is 30 positions long between 43nd and 72nd positions in the sequence to help with FAD binding to catalyze the oxidation of glucose into hydrogen peroxide and D-Glucono- δ -lactone. Another helpful feature is that the isoelectric point is 4.22 at physiological pH (Uniprot, 2016). Since glucose oxidase has an extremely negative charge on its phosphate backbone a phosphate buffered solution (PBS) with additional Mg2+ is required to combat the negative charge. This buffer with additional bovine serum albumin (BSA) and sodium azide will allow glucose oxidase to remain stable for over 2 years if kept at 4oC (MegaEnzyme, 2014).

At this time, no viable results have been found. This selection process is almost through with Round 2 and is currently undergoing large scale PCR. At the conclusion of Round 1 there was 1228.2 ng/µl of RNA present in the eluted RNA. The main point of this selection process is to find an aptamer that will bind to glucose oxidase for an ELOSA in order to detect abnormal increase of proislet amyloid polypeptide using a colorimetric response. By detecting this increase, early detection of diabetes type 2 mellitus can occur.

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