Jiae An's GOx Aptamer Selection (2016)

Galactosemia is an uncommon inherited disorder that affects the metabolism of galactose. A small amount of galactose is present in many foods. It is primarily part of a larger sugar called lactose, which is found in all dairy products and many baby formulas. The signs and symptoms of galactosemia results from an inability to use galactose to produce energy. According to the Galactosemia Foundation, classic galactosemia occurs in 1 in 30,000 to 60,000 newborns (Elsas LJ , 2005). Since this disorder is also inherited from parents, mutations occurred in a specific gene such as GALT, GALKI, and GALE cause galactosemia. These genes are in charge of providing instructions for making enzymes that are essential for processing galactose obtained from the diet. These enzymes break down galactose into another simple sugar, glucose, and other molecules that the body can store or use for energy. Since this uncommon disorder is imperative to be diagnosed during the early age of life, a better diagnostic technique, less harmful, is required for babies before they consume milk.

Enzyme linked immunosorbent assay (ELISA) method can be used to diagnose galactosemia. This technique was developed in 1971 to replace the radioimmunoassay (Toh S. et al. 2014). This consists of a target, antibody and a detection that brings out the signal when detected the antigen. An aptamer can replace the antibody used in ELISA because it is more secured and unhazardous.

An aptamer is a nucleic acid that binds targets with specificity and a high affinity. These short pieces of DNA or RNA bind to a molecular target such as a protein or a small molecule. An aptamer is very similar to antibodies that function as a binding specie, detecting molecule and inhibiting specific target. Aptamer utilizes in vitro selection that aptamers are selected with the unbound DNA being filtered out, leaving the only bound DNA. An aptamer has a characteristic of tight binding specificity to the specific target molecule whether it is a protein or a small molecule with amino acid sequence (Stoltenhurg, R, et al 2007) .

Glucose oxidase is a small, stable enzyme that oxidizes glucose into gluconic acid, producing hydrogen peroxide from oxygen. Since hydrogen peroxide is a toxic, it helps to protect against bacterial infection. As shown in the figure 2., it consists of two subunits each containing 80kDa each, weighing 160kDa total (Raba J, et al. 1995).

In the ELISA method, as shown in figure 3, an aptamer for glucose oxidase can bind for a particular antigen that detects the sign of galactosemia for babies. Aptamer can be attached to the glucose oxidase once developed to serve as a reporter molecule inducing a chromatic change when detecting the positive result in disorder such as galactosemia.

Throughout the course of research history, an aptamer selection for glucose oxidase has not been found. However, developing an aptamer for glucose oxidase would contribute to diagnose sugar-related more effectively. A RNA aptamer selection against glucose oxidase will be performed for the development of a possible aptamer to replace the antibody that acted as a reporter molecule of galactosemia that affects the metabolism of sugar in early stage of life.

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References

Elsas LJ. (2000, 2005). “Galactosemia. GeneReviews, University of Washington,”

Molecular Explorations through biology and Medicine (2006). Picture retrieved from http://pdb101.rcsb.org/motm/77

Pazur, J. H., and Kleppe, K., (1964) “The oxidation of glucose and related compounds by glucose oxidase from Aspergillus niger. Biochemistry”, 3, 578-583.

Swoboda, B. E. P., and Massey, V., (1965) “Purification and properties of glucose oxidase from Aspergillus niger. J. Biol. Chem.”, 240, 2209-2215

Stoltenhurg, R., C. Reinemann, and B. Strehlitz. (2007). “SELEX—a (r)evolutionay method to generate high-affinity nucleic acid ligands”. Biomol Eng. 381-403

Toh, S., Citartan, M., Gopinath, S., and Tang, H. (2014). “Aptamers as a replacement for antibodies in Enzyme-linked Immunosorbent Assay” Elsevier (64). 392-403

Raba, J and Mottola, H. (1995). “Glucose Oxidase as an Analytical Reagent. Critical Reviews in Analytical Chemistry” 25 (1): 1–42