Alexandra Miller's GOx Aptamer Project (2016)

RNA Aptamer Selection Against Glucose Oxidase for Detection of Bacterial Meningitis During Colonization of the Nasopharyngeal Cavity

Introduction/Background

Since it’s first detection by Vieusseux in 1806, bacterial meningitis has been considered almost uniformly fatal, and continues to have an unacceptably high morbidity rate despite the development of antimicrobial agents and diagnostic techniques. Epidemic meningitis primarily affects regions with limited resources. The epidemic in 1996 in the “meningitis belt” of sub-Saharan Africa resulted in over 300,000 cases with 30,000 deaths (Tikhomirov; Santamaria; Esteves 1997). Approximately 1.2 million cases of bacterial meningitis occur annually worldwide with 135,000 deaths (World Health Organization, 1998). The further development of cost-effective and reliable diagnostic and therapeutic technologies will be essential in moderating the spread of the disease, particularly in areas such as sub-Saharan Africa.

N. meningitidis, a gram-negative diplococcus human pathogen, first colonizes of the nasopharynx and can lie dormant there (Scheld; Uwe Koedel; Barnett; Pfister, 2002). This phase of the infection occurs before migration into the intravascular and subarachnoid spaces. The embedded protein Neisseria hia/hsf homolog A (NhhA) shown in blue in Figure 1 attaches the bacteria to the body’s endothelial cells during hibernation. This experiment endeavors to develop a diagnostic technique that will signal the presence and concentration of the bacteria in the mucous membranes of the nasopharynx by binding to NhhA (Sjölinder; Altenbacher; Hagner; Sjölinder, 2012).

GOx is a highly stable enzyme that catalyzes the oxidation of β-D-glucose into D-glucono-1,5-lactone, which then hyrdolyzes to gluconic acid and converts O2 to H2O2 as shown in Figure 2 (Keilin; Hartree, 1952). This causes a color change that can be recorded using spectrophotometry, making GOx an optimal reporter molecule.

GOx is a glycoprotein containing 16% neutral sugar and 2% amino sugars, and can be found naturally in various species of fungi and insects such as Asperfillis niger and the Honey Bee, as well as in human blood (Goodsell, 2006). GOx is a 160 kDa dimer (2 subunits of 80 kDA each) with pH 5.41, isoelectric point of 4.2, and an estimated charge of -26.2. It functions optimally at a pH of 7.3 and in the presence of the cofactor flavin adenine dinucleotide (FAD) as shown in Figure 3 (Bhatti, 2006).

GOx is commonly used as a reporter molecule in glucometers, which measure the concentration of H2O2 produced during exposure of blood to glucose oxidase estimating the blood sugar in diabetic patients. Researchers of the The Center of Nanoscience and Nanotechnology at The Hebrew University of Jerusalem have used GOx to detect the presence of aptamer-throbin complexes (Golub; Freeman; Niazov; Willner, 2011).

Bead-based selection, shown in Figure 4 selects oligonucleotide sequences, called aptamers, through the separation of nonbinding and binding species and enrichment of the bound sequences. These sequences have a high affinity for a target (ion, peptide, protein, etc.) given selected conditions. They are highly specific, cost effective, reliable, and have downstream applications in therapeutics, diagnostics, and drug delivery. The goal of this experiment is to find and enrich an aptamer for GOx that will detect bacterial meningitis caused by N. meningitides during the colonization of the nasopharyngeal cavity. This will be accomplished using an aptamer for Glucose Oxidase that conjoins with an aptamer that binds to NhhA.

The aptamer complex will detect the presence and concentration of the bacteria in the following method: expose to the NhhA at 25°C, separate using centrifugation, remove the supernatant containing unbound glucose oxidase and aptamers, resuspend in buffer containing β-D-glucopyranose and in O2 rich environment, then detect the resulting color change using spectrophotometry or detect the increase in H2O2 using a platinum electrode. The approach using the glucometer would be much more portable and efficient in resource limited regions.

An RNA aptamer selection is underway. Once an aptamer has been selected for and a conjugate aptamer has been selected for NhhA, an affordable, portable, and simple diagnostic tool can be developed for bacterial meningitis.

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References