Marco Munoz's GOx Aptamer Project (2019)

Anti-Nanoluciferase Aptamer to Improve Urine Glucometers

Introduction and Background:

The purpose of this experiment is to find an inhibitory aptamer to the target nanoluciferase, a bioluminescent reporter molecule, for a diagnostic application for diabetes using a His-Tag Bead-ased SELEX method. An aptamer is an oligonucleotide (RNA or DNA sequence) that binds to a target protein with high affinity in specific predetermined conditions. This selection method will bind the target protein to the beads to allow for the most strongly bound sequences of the N71 RNA pool to bind to the target by washing off weak sequences with MES buffer. The target of this lab is nanoluciferase, which is a bioluminescent protein found in deep sea shrimp. Nanoluciferase is a reporter molecule that is cheaper to produce than most other luminescent proteins due to its very stable structure that allows it to even withstand extremely high temperatures for an extended period of time. An added benefit is that it will glow a hundred times brighter than other bioluminescence such as fireflies (“Nanoluc®,” 2019). An aptamer for nanoluciferase would improve urine tests for glucose in an effort to make them more affordable and available for diabetics. This would be done using an ELONA diagnostic assay. An ELONA diagnostic assay in aptamer research is when two different aptamers are covelantly bonded together by their tails which leaves their binding site available to bind to their respective proteins. In this case, the Anti-Nanoluciferase Aptamer would be engineered to be linked to a Glucose Aptamer so that in the presence of glucose, the aptamers would unfold, allowing Nanoluciferase to luminesce (Figure 1).

There are many problems with current urine glucometers that could be improved on with this potential aptamer. Current urine glucometers tests are difficult to read and provide false positives often (Figure 2). These false positives could be due to many factors including possible medications the patient is on or something as simple as checking the results on the test just seconds too late (“Diabetes and urine,” 2019). This could theoretically be fixed by the implementation of my aptamer due to its high specificity and durability. Knowing glucose levels in urine is an important step in the health care of diabetics as it can provide vital information on kidney health.

The goal of the ELONA diagnostic assay would be to engineer a glucose aptamer to bind to the anti-nanoluciferase aptamer so that in the presence of glucose, the inhibitory nanoluciferase aptamer will denature and allow nanoluciferase to naturally produce luminescence. The amount and intensity of luminescence could then be measured to proportionally measure the amount of glucose in the urine as a single glucose molecule would activate a single nanoluciferase.

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References

Diabetes and urine glucose monitoring. (2019, April 18). Retrieved from https://www.mydr.com.au/tests-investigations/diabetes-and-urine-glucose-monitoring

NanoLuc® Luciferase: One Enzyme, Endless Capabilities. (n.d.). Retrieved from https://www.promega.com/resources/technologies/nanoluc-luciferase-one-enzyme-endless-capabilities/

New CDC report: More than 100 million Americans have diabetes or prediabetes | CDC Online Newsroom | CDC. (n.d.). Retrieved from https://www.cdc.gov/media/releases/2017/p0718-diabetes-report.html

Wu, Y., Kwon, Y. (2016) Aptamers: The “evolution” of SELEX. Methods, 106 21-28.