Ted Shi's GOx Aptamer Project (2016)

Due to the stresses that hospitalization and perioperative care can have on the body, there are many times when patients themselves need help obtaining the nutrients they need to survive and recuperate. (Martindale et al. 2013) Although many methods have been devised to counter the effect of malnutrition in these cases, one common and straight-forward solution is enteral tube feeding (ETF) (National Collaborating Centre for Acute Care 2006).

Enteral tube feeding (Figure 1) can be implemented through the nasopharynx, oropharynx, duodenum, or jejunum, depending on the condition of the gastrointestinal tract. By delivering fluid-containing nutrients straight into the body, the process bypasses the need for voluntary masticatory processes. This is especially useful when the patient has been unconscious or has severely weakened muscles for an extended amount of time.

Feeding tubes can be inserted through the nose and slid down to the stomach.

However, one of the most common problems with enteral tube feeding is the possibility of the patient aspirating the nutritive solution. There can be issues with the tube that, during long periods of insertion, may cause it to leak. The fluid, which then proceeds down the trachea and end up in lungs, may cause infections that further worsen the condition of the patient. Aspiration pneumonia, a symptom of this issue, comprise of 5% to 15% of the total pneumonia cases in hospitals and has caused up to 30% of anesthesia procedure deaths in longitudinal studies (Ufberg et al. 2005). Though it is possible to treat these infections with antibiotics, it is much more important to prevent such infections from happening in the first place.

Furthermore, current testing technologies for possible aspiration have shown substandard performance results. Earlier tests involving blue dye mixed into the nutritive fluid and an accompanying glucometer found poor results; visual inspections of the tracheal secretions for the presence of the same blue dye were much less sensitive than a measurement by a glucose test strip or a glucometer (Potts et al.1993). More recent pepsin assays have as low as 20%, and as high as 50%, chance of correctly identifying if a patient has aspirated during intubation or while being intubated (Bushra et al. 2002, Ufberg et al. 2005).

Glucose oxidase (Figure 2), an enzyme used in a multitude of industries (Wong 2008), may serve to alleviate our fluid aspiration problem. The enzyme, comprised of two primers weighing 80 kDa each for a total of 160 kDa, is most commonly found as an antibiotic in fungal and insectoid organisms. In more recent years, the cultivation of better proteins by the Aspergillus niger fungus have increased its usability in all contexts (Bhatti 2006). In our context, the protein functions to convert available glucose to hydrogen peroxide (Figure 3).

The proposed solution to aspiration detection is a new enteral feeding tube (Figure 4). The barrier layer, an integral part of the tube, will house the buffered enzyme-aptamer solution. Potential leaks in the innermost tube will introduce the simple sugars to the glucose oxidase solution in the barrier layer, where glucose oxidase, as a reporter molecule, will be able to convert simple sugars in the nutritive fluid into hydrogen peroxide. Through an attachment of a metal wire, the hydrogen peroxide could be used to change the voltage of the wire, and signal to healthcare providers whether the fluid has leaked and whether to change the tube.

Aptamers are short oligonucleotide molecules that are able to bind to larger targets, such as the glucose oxidase enzyme. Through the process of aptamer selection, the pool of RNA can be narrowed down and then amplified. Aptamers, made from nucleic acids, are more versatile than antibodies in many ways. They are able to refold themselves after denaturation and are very specific in their binding to protein targets. This makes it especially useful in our scenario, as the aptamer solution will be exposed to a wide variety of environments, from a cold operating room, to a relatively lukewarm post-anesthesia care unit ward, to a variable hospital room. Either way, the aptamer’s versatility is crucial in its function of glucose oxidase.

The Systematic Evolution of Ligands by Exponential Enrichment, or SELEX, is the method through which the aptamers that bind with a high specificity to the reporter enzyme are found. Aptamer selection begins with a large pool of around 1012 to 1014 different sequences of RNA. The N71 pool utilized in this experiment contained a random region of 71 nucleotides, surrounded by constant regions needed for recognition for other enzymatic processes (Bell et al. 1998). The binding process uses M270 Streptavidin Nanobeads to affix the protein target through which magnets can select for bound aptamers after incubation. The RNA sequences that do bind are then run through reverse transcription to create DNA and amplified through polymerase chain reactions (PCR). Amplified sequences will then be run through transcription to be turned back into RNA and then purified through polyacrylamide gel electrophoresis and ethanol precipitation. This enriched pool, consisting of significantly fewer variation in sequences, is then used for future rounds of selection.

However, there have already been aptamers developed for glucose oxidase. Mostly used in the sense of biomolecular sensors, much of the research is still in-progress. In this project, a stabilizing aptamer complex, attached to glucose oxidase, will serve as an inhibitor until the enzyme is ready to be used. By stabilizing the glucose oxidase in a variety of environments outside of where it is needed, we can lengthen its shelf life as well as increase its accessibility to consumers. By selecting the aptamer in physiological conditions, we can consider the downstream application of the aptamer when it is used in nasogastric ETF.

Though we do not yet know whether the N71 pool aptamer will attach to the active site of the enzyme or to an allopatric site, there are two possibilities for an aptamer of this nature. The easier of the two - an aptamer binding to the allopatric site - allows for the glucose oxidase to function correctly when injected as a solution into the barrier layer. If the aptamer were to bind to the active site of the enzyme, and thus restrict its function, we would need a way for the aptamer to detach at physiological temperature to allow glucose oxidase to function.

A RNA aptamer selection is underway for this project, and I hope to see research centered on glucose oxidase and its ability as a reporter enzyme. So far, only 7 rounds of selection have been performed against the target. Sequencing and a binding assay have also been done to identify whether or not an aptamer is present. Ultimately, this project will find an aptamer that will stabilize and inhibit glucose oxidase and detach when the enzyme is needed.

Click here for the Final Report

References

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Bell, S.D., Denus, J.M., Dixon, J.E., and Ellington, A.D. 1998. RNA molecules that bind to and inhibit the active site of tyrosine phosphatase. The Journal of Biological Chemistry 273:14309-14314.

Bhatti, H.N., Madeeha, M., Asgher, M. and Batool, N. 2006. Purification and thermodynamic characterization of Glucose Oxidase from a newly isolated strain of Aspergillus niger. Canadian Journal of Microbiology 52:519-524.

Bushra, J.S., Ufberg, J.W,. Karras, D.J., and Kueppers, F. 2002. Incidence of aspiration after emergency endotracheal intubation and association with clinical suspicion. Academic Emergency Medicine 9:405.

Martindale, R.G., McClave, S.A., Taylor, B., and Lawson, C.M. 2013. Perioperative nutrition: what is the current landscape? Surgical Nutrition Summit Reports 37(1):5-20.

National Collaborating Centre for Acute Care. Nutrition support for adults: oral nutrition support, enteral tube feeding and parenteral nutrition: methods, evidence & guidance. London: National Collaborating Centre for Acute Care, at the Royal College of Surgeons of England, 2006. Print.

Potts, R.G., Zaroukian, M.H., Guerrero, P.A., and Baker, C.D. 1993. Comparison of blue dye visualization and glucose oxidase test strip methods for detecting pulmonary aspiration of enteral feedings in intubated adults. Chest 103:117-121.

Shlyahovsky, B., Li, D., Katz, E., and Willner, I. 2007. Proteins modified with DNAzymes or aptamers act as biosensors or biosensor labels. Biosensors and Bioelectronics 22:2570-2576.

Sarmaga, D., DuBois, J.A., and Lyon, M.E. 2011. Evaluation of different disinfectants on the performance of an on-meter dosed amperometric Glucose-Oxidase-Based glucose meter. Journal of Diabetes Science and Technology 5:1449-1452.

Ufberg, J.W., Bushra, J.S., Karras, D.J., Satz, W.A., and Kueppers, F. 2005. Aspiration of gastric contents: association with prehospital intubation. American Journal of Emergency Medicine 23:379-82.

Wong, C.M., Wong, K.H, and Chen, X.D. 2008. Glucose oxidase: natural occurrence, function, properties and industrial applications. Applied Microbiology and Biotechnology 78:927-938.