Alec Agha's GOx Aptamer Selection (2016)

Use of Aptamers with Glucose Oxidase as a Biofuel

Introduction/ Background

Keeping in mind that people are constantly supplying their own bodies with the power source that are carbohydrates, it becomes intuitive to do the same with any type of electronic device that works within a person’s body. Glucose oxidase, among other oxidation enzymes, bridges this gap specifically between glucose and electronic power particularly well, with capabilities of being generally implemented in several battery-operated bio-electric devices such as pacemakers or implantable cardioverter defibrillators, among many other things.

Although enzymatic batteries seem like a reasonable alternative to conventional batteries, when speaking in terms of an in vivo power source, there comes several fallbacks withholding implementation. The main issue that is to be addressed in this research is within the fact that naturally occurring enzymes do not have a long lifespan. Subsequently, although enzymes like GOx produce a large amount of electrons in their reaction mechanism, it becomes difficult removing these electrons from the enzyme to produce a current. Additionally, enzymes normally float freely within a cell, whereas in this case they must be immobilized to form a capable cathode and anode (Atanassov et al., 2007). Several of these aforementioned problems are currently being addressed and solved however; for example, the use of redox mediators to accept electrons away from whichever enzyme is in question (Ramanavicius et al., 2015), as well as hydrophobically modified micellar polymers to form a stable microenvironment for efficient immobilization onto an anode/cathode (Atanassov et al., 2007). The main issue of enzymatic lifespan too seeks solution, and in this case, via the use of aptamers.

Glucose oxidase plays a key role in among several other industrial applications as a catalase for the oxidation of β-d-glucose to gluconic acid along with the simultaneous production of hydrogen peroxide (Fig. 2). In a battery system, GOx will work as the anode end of the voltaic cell in its oxidation of glucose, creating a maximum theoretical electromotive force (emf) of 1 V (Bankar et al., 2009). The simultaneous production of hydrogen peroxide (H2O2) in this redox reaction is notable in the case of in vivo application, as H2O2 is clearly toxic in human bloodstream; instead this by-product can be used to fuel the subsequent cathode end of the cell via oxygen or hydrogen peroxide utilizing enzymes, particularly horseradish perodixase (HRP). This enzyme coupled system can best be seen in figure 1.

GOx itself, derived from the fungus Aspergillus niger, is a 160 kDa glycoprotein dimer comprised of two identical subunits. What is notable in its structure are the two FAD coenzymes bound: the electron acceptors that are the source of GOx’s amperometric ability (Wong et al., 2008), which is to be exploited in an enzyme-battery system. These FAD coenzymes show a promising ability of nucleic acid species to already be able to bind to GOx, such as aptamers (Fig. 3), giving rise to an inhibitory interest.

Aptamers are oligonucleotides, in this case RNA, with regions of random nucleotide sequence that allow for high specificity. This specificity, in addition to their small size and stability, give rise to unique binding affinity for a given target, which introduce a variety of application. Drug delivery, specifically in the use of nanoparticles, can be used in conjunction with aptamers to produce increased targeting and more efficient therapeutics (Levy-Nissenbaum et al., 2008). Aptamers can furthermore be used as an effective inhibitor for a given protein’s function, or as a form of diagnostic in detecting a target’s presence. In the case of GOx, aptamers are selected to inhibit the protein’s function, which is beneficial in preserving GOx while its operating device is not in use. In the case that the bio-electric device is in use, the aptamers can be removed via RNAse, pH, voltage or temperature change, to restore functionality to GOx and to the glucose reaction. Degradation of this protein, proven to be problematic in its use in glucometers for example, concerning glucometer accuracy (Tonyushkina, 2009), is limited through this type of deactivation and reactivation.

Glucose oxidase, while having suitable nucleotide binding sites because of its FAD coenzymes, is in contrast largely electronegative, making negatively charged RNA aptamers particularly difficult to bind, possibly providing reason why no aptamer has been developed for this target. No particular buffer must be used with this protein, although due to its overall negativity, the addition of divalent salts (e.g. MgCl2) is necessary. Because the primary application of this aptamer is to be used within the human bloodstream however, buffers (e.g. PBE) similar to physiological conditions are used.

With these specific factors working for and against aptamer-binding potential, and using the following procedures and parameters, which will be specified more in detail, results so far are only marginal, again with only one full round of selection having been performed as of now. This first round pool of aptamers, using sequencing, do not completely show any major signs of selection occurring, and thus cannot effectively inhibit GOx’s redox function as of yet. After subsequent rounds of selection and enrichment, GOx inhibition should become statistically visible through a binding assay. Once this problem is addressed, so too will the lifespan problem of GOx be effectively addressed in created a better, longer lasting enzyme capable of replacing standard bio-electric power sources.

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References

Atanassov, Plamen et al. “Enzymatic Biofuel Cells.” Interface-Electrochemical Society 16.2 (2007): 28–31. Print.

Bankar, Sandip B. et al. “Glucose Oxidase — An Overview.” Biotechnology Advances 27.4 (2009): 489–501. ScienceDirect. Web.

Goodsell, D.S. “Glucose Oxidase.” RCSB Protein Data Bank (2006): n. pag. CrossRef. Web. 19 Sept. 2016.

Levy-Nissenbaum, Etgar et al. “Nanotechnology and Aptamers: Applications in Drug Delivery.” Trends in Biotechnology 26.8 (2008): 442–449. ScienceDirect. Web.

Ramanavicius, Arunas et al. “Biofuel Cell Based on Glucose Oxidase from Penicillium

3333333333Funiculosum 46.1 and Horseradish Peroxidase.” Chemical Engineering Journal 264 (2015): 165–173. ScienceDirect. Web.

Tonyushkina, Ksenia, and James H. Nichols. “Glucose Meters: A Review of Technical Challenges to Obtaining Accurate Results.” Journal of diabetes science and technology (Online) 3.4 (2009): 971–980. Print.

Witt, S et al. “Conserved Arginine-516 of Penicillium Amagasakiense Glucose Oxidase Is Essential for the Efficient Binding of Beta-D-Glucose.” Biochemical Journal 347.Pt 2 (2000): 553–559. Print.

Wong, Chun Ming, Kwun Hei Wong, and Xiao Dong Chen. “Glucose Oxidase: Natural Occurrence, Function, Properties and Industrial Applications.” Applied Microbiology and Biotechnology 78.6 (2008): 927–938. CrossRef. Web.