Peter Mai's GOx Aptamer Selection Project (2019)

Tethering Inhibitory Aptamers to Glucose Oxidase for Accessible Glucose Monitoring Diagnostics

Introduction and Background

According to the International Diabetes Federation, 79% of adults with diabetes are living in low and middle-income countries. The prevalence of diabetes, especially in low-income counties, is a cause for concern because diabetes has caused millions of deaths, many of which are linked to the lack of affordable, accessible, and accurate glucose diagnostics. Current glucose diagnostic solutions involve the use of a single-use unstable GOx test-strips to monitor glucose levels. Aptamers have the potential to stabilize and extend the life of GOx. Aptamers are molecules that have a binding affinity to a target and are created through selection from large sequence pools (Lakhin et al. 2013). In comparison to antibodies, Aptamers are more versatile due to their higher binding affinity and cost efficiency (Hidding n.d.).

Understanding the function of GOx is the key to developing an aptamer solution. Glucose oxidase (GOx) is an enzyme which catalyzes the oxidation of glucose to hydrogen peroxide, providing current glucometers the ability to measure glucose levels indirectly (Figure 1) through measuring the concentration of hydrogen peroxide produced when current is applied to a test strip (Wong et al. 2008). While current solutions for glucose monitoring are adequate for single-use cases, this method proves to be expensive, as glucose oxidase loses its function after a current is applied, and glucose levels are measured. An inhibitory aptamer for glucose oxidase offers a potential solution to inhibit the loss of GOx function and allow for continuous monitoring of glucose levels without replacing the GOx in test strips. This would allow for glucose oxidase to be placed into the glucometer itself instead of the test strips, lowing cost by requiring only the test strips to be replaced, and not GOx.

To find an aptamer for GOx, filter-based in vitro selection will be performed through the SELEX method (Figure 2). Throughout the SELEX process, RNA from the N71 pool that has high specificity will bind to GOx and be converted to DNA, amplified, converted back to RNA, and isolated and purified in a PAGE gel. Through multiple iterations of the SELEX process, more specific molecules that bind to the target will be isolated. Ultimately, this process, an inhibitory aptamer will potentially be discovered to inhibit glucose oxidase in glucose test strips from degrading and allow enhanced accuracy and versatility of glucose monitoring.

Progress has been made, and two rounds of selection has been completed. An analysis of an lsPCR revealed bands of the expected length with proper amplification. A visualization of the PAGE gel reveals a visible shadow, which confirmed a successful page and RNA purification. A successful round of selection has now been completed, and this is positive progress towards the development of an inhibitory aptamer. The main objective for performing the SELEX method is to find an inhibitory aptamer for glucose oxidase. After multiple rounds of selection, the RNA pool will be assayed for binding affinity to the target, and positive results would suggest the presence of aptamer candidates. This will further the progress of developing an inhibitory aptamer for GOx. This will ultimately inhibit glucose oxidase in glucose test strips from degrading and allow enhanced accuracy, versatility, and affordability for glucose monitoring.

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References

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