Basmah Barkatullah's GUS aptamer project (2016)

Jaundice is one the most common conditions affecting newborns. Jaundice causes discoloration of the infant’s skin and whites of the eyes (see figure 2) and in extreme cases large bilirubin buildup can cause brain damage. An excess of β-glucuronidase in breast milk is associated with an infant developing jaundice.

β-glucuronidase is found in humans, many animals, and in bacteria such as Escherichia coli,4 and is localized in the lysosome 5. It is a tetramer with a molecular weight of 57,000 g/mol.

This enzyme is expressed in most tissues in humans, but plays an important role in the small intestine. Specifically, β-glucuronidase deconjugates bilirubin glucuronide into free bilirubin and glucuronic acid5. Free bilirubin can then be released back into the bloodstream, which can cause jaundice1. Theraputic research on β-glucuronidase has focused on mutations in the gene coding for the production of this enzyme which result in a of β-glucuronidase deficiency. This leads to a buildup of glycosaminoglycan in the lysosome which can be lethal 5. Research on injecting this enzyme into the body to cure this deficiency has been a popular area of research. Sands et al. injected β-glucuronidase that had been produced recombinantly 6 into mice lacking β-glucuronidase and found that it helped treat the symptoms of β-glucuronidase deficiency 5.

The goal of this project is to find an aptamer for β-glucuronidase. Aptamers are nucleic acid species that bind with high affinity to a certain molecule. They have the ability to bind to a wide variety of molecules and can be used in therapeutics, diagnostics, drug delivery and systems biology applications. Aptamers are in general much more cost-effective and efficient to produce than antibodies. The aptamer found for β-glucuronidase will be used as part of a diagnostic tool for breast milk jaundice and will also replace an antibody.

In this capture ELISA, β-glucuronidase from a sample of breast milk is immobilized by an antibody. The aptamer will then bind to the β-glucuronidase which will allow the reporter molecule to act on its substrate and allow for the detection of unhealthy, high levels of β-glucuronidase in breast milk.

Aptamers are generated using the aptamer selection process. It is a cycle involving six steps: target immobilization, target and RNA pool incubation, separation of bound and unbound RNA, elution of RNA, concentration of RNA, and amplification of RNA pool so that it can be used to repeat the cycle. This process repeats for multiple rounds of selection until an aptamer with a specific sequence that binds with high affinity to the target is produced. The target, β-glucuronidase, has a His tag which means nickel beads will be used to immobilize the target. However, β-glucuronidase is also a difficult target to produce and aptamer against because it is not known to bind nucleic acids, does not have a binding pocket, and has a negative charge of -21.2 at pH 7.4. Since it is negatively charged, the selection buffer will need extra magnesium chloride to help the RNA bind to the target. The β-glucuronidase is stored in PBS buffer and this buffer can also be used for the selection since breast milk is around pH 7.4, however extra magnesium chloride will still be needed 7. An aptamer against β-glucuronidase does exist, but it has not been used in any applications 8.

Overall, the goal of this project is to find an aptamer that binds to β-glucuronidase in breast milk that could be used as a diagnostic tool for breast milk jaundice. The first round of selection has been started and large scale PCR is underway to amplify the species that bound to the target. With enough rounds, specificity of the RNA species to the target will increase.

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References

1. Thor WR Hansen (2016). Neonatal Jaundice: Background, Pathophysiology, Etiology. Available at: http://emedicine.medscape.com/article/974786-overview#a5

2. Huma Naz, A.I., Waheed, A., Ahmad, F., Sly, W., and Hassan, I. (2013). Human β -Glucuronidase: Structure, Function, and Application in Enzyme Replacement Therapy. Rejuvenation Research 16.

3. Prashant G Deshpande (2016). Breast Milk Jaundice: Background, Pathophysiology, Epidemiology. Available at: http://emedicine.medscape.com/article/973629-overview#a5.

4. Skar, V., Skar, A.G., and Strømme, J.H. (1988). Beta-glucuronidase activity related to bacterial growth in common bile duct bile in gallstone patients. Scand. J. Gastroenterol. 23, 83–90.

5. Vogler, C., Sands, M.S., Levy, B., Galvin, N., Birkenmeier, E.H., and Sly, W.S. (1996). Enzyme Replacement with Recombinant β-Glucuronidase in Murine Mucopolysaccharidosis Type VII: Impact of Therapy during the First Six Weeks of Life on Subsequent Lysosomal Storage, Growth, and Survival. Pediatr Res 39, 1050–1054.

6. Sands, M.S., Vogler, C., Kyle, J.W., Grubb, J.H., Levy, B., Galvin, N., Sly, W.S., and Birkenmeier, E.H. (1994). Enzyme replacement therapy for murine mucopolysaccharidosis type VII. Journal of Clinical Investigation 93, 2324–2331.

7. Malhotra, S.L. (1982). Effect of non-suckling on the pH of breast milk and its possible relationship with breast cancer. Postgrad Med J 58, 749–752.

8. Qiao, L., Lv, B., Feng, X., and Li, C. (2015). A new application of aptamer: One-step purification and immobilization of enzyme from cell lysates for biocatalysis. Journal of Biotechnology 203, 68–76.

9. Jaundice-In-Newborn-Babies.jpg (JPEG Image, 420 × 420 pixels). New Kids-Center Available at: http://www.newkidscenter.com/images/10415990/Jaundice-In-Newborn-Babies.jpg.

10. Malhotra, S.L. (1982). Effect of non-suckling on the pH of breast milk and its possible relationship with breast cancer. Postgrad Med J 58, 749–752.