Doru Gucer's HRP Aptamer Project (2019)

ELONA based Diagnostic Tool for Alzheimer’s Disease utilizing an Anti-Horseradish Peroxidase Detection Aptamer

Introduction and Background:

Despite the significant medical and economic burdens Alzheimer’s Disease has had on populations around the world, a reliable method of diagnosing the onset of the disease remains out of reach (Perl, 2010). The merit of early diagnoses may not be immediately evident as Alzheimer’s remains an irreversible and incurable condition. However, when one considers current FDA approved methods of slowing the progression of the disease, one of which has been found to reduce average medical costs by $3,891 compared to a control group, the immediate benefit of developing such a tool is shown (Leifer, 2003).

Although much remains to be known regarding the mechanisms of Alzheimer’s, it has been widely accepted that Alzheimer’s is largely characterized by an overabundance of senile plaques (Giannakopoulos, Hof, Michel, Guimon, & Bouras, 1997). Although such plaques are also found in normal aging brains, they are found to be overexpressed in patients suffering from Alzheimer’s. Composed of an amyloid core, senile plaques involved in the early development of Alzheimer’s are largely dependent on β-amyloid peptide production. This production has been found to be facilitated by BACE1, an enzyme with a B1-CT binding tail that cleaves the amyloid precursor protein (Rentmeister, 2006). Therefore, increased levels of BACE1 in the brain could be indicative of increased senile plaque development found in Alzheimer’s.

Thus, the downstream application of this study will be comprised of forming an ELONA with a detection aptamer bound to Horseradish Peroxidase (HRP) and a capturing aptamer immobilizing the structure on a glass plate, as seen and described in Figure 1, to detect elevated levels of BACE1 in the brain, with the hopes of being able to diagnose Alzheimer’s (ELONA, 2017). HRP is a glycoprotein that has previously played major roles in diagnostic assays due to its fluorescing capabilities and is prized for its relative stability, cost effectiveness, and high specificity to its targets, especially when compared to competing enzymes like Alkaline Phosphatase (Beyzavi, K. et. Al, 1987). In 2006, a group from the University of Bonn succeeded in identifying an aptamer that could serve as the capturing aptamer in the proposed assay by binding to this B1-CT site, (Rentmeister, 2006). As a result, selecting for a detection aptamer specific to HRP in this ELONA structure could result in the ability to detect the elevated BACE1 levels in a target area, serving as a reliable method of diagnosing the onset of Alzheimer’s disease.

Although there are several different methods to bind pools of genetic material to targets, a beadbased SELEX method was used for the purposes of this aptamer selection, as described in Figure 2 (Fraser, 2017). The HRP target in the study was biotinylated; therefore, streptavidin beads were the bead selection method of choice, considering their binding affinity to biotin. A PBS selection buffer was used due to its pH being close to that of blood at 7.4 while the binding reaction took place at 37 degrees Celsius to simulate body temperature. Unbound RNA was discarded, and bound RNA was amplified and purified for use in future rounds of selection using reverse transcription, ccPCR, lsPCR, transcription, and PAGE processes. Upon isolating a viable aptamer sequence, an ELONA will be formed with the University of Bonn’s capturing aptamer. This complex will serve as diagnostic tool for the abnormal production of β-amyloid peptides as the isolated detection aptamer will induce fluorescence in HRP whenever the capturing aptamer binds to a BACE1 B1-CT site.

Round 2 is where the present state of aptamer selection is. Upon completing a PAGE gel, and isolating template for the next round, Round 3 of aptamer selection will begin.

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References:

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ELONA: The evolution of ELISA. (2017, September 05). Retrieved April 15, 2019, from https://www.aptamergroup.co.uk/elona-evolution-elisa/

Fraser, L., Liang, S., Shiu, S. C.-C., & Tanner, J. (2017). Aptamer Bioinformatics. International Journal of Molecular Sciences, 18, 2516. https://doi.org/10.3390/ijms18122516

Giannakopoulos, P., Hof, P. R., Michel, J.-P., Guimon, J., & Bouras, C. (1997). Cerebral cortex pathology in aging and Alzheimer’s disease: A quantitative survey of large hospitalbased geriatric and psychiatric cohorts. Brain Research Reviews, 25(2), 217–245. https://doi.org/10.1016/S0165-0173(97)00023-4

Leifer, B. P. (2003). Early Diagnosis of Alzheimer’s Disease: Clinical and Economic Benefits. Journal of the American Geriatrics Society, 51(5s2), S281–S288. https://doi.org/10.1046/j.1532-5415.5153.x

Murphy MP, LeVine H 3rd. Alzheimer's disease and the amyloid-beta peptide. J Alzheimers Dis. 2010;19(1):311–323. doi:10.3233/JAD-2010-1221

Perl DP. Neuropathology of Alzheimer's disease. Mt Sinai J Med. 2010;77(1):32–42. doi:10.1002/msj.20157

Rentmeister, A. RNA aptamers selectively modulate protein recruitment to the cytoplasmic domain of -secretase BACE1 in vitro. RNA Society, 2006, 12(9), 1650- 1660. doi:10.1261/rna.126306

Veitch, N. C. Horseradish peroxidase: A modern view of a classic enzyme. Phytochemistry, 2004, 65(3), 249-259. doi:10.1016/j.phytochem.2003.10.022

Xiang, J., Zhang, W., Cai, X.-F., Cai, M., Yu, Z.-H., Yang, F., … Cai, D.-F. (2019). DNA Aptamers Targeting BACE1 Reduce Amyloid Levels and Rescue Neuronal Deficiency in Cultured Cells. Molecular Therapy - Nucleic Acids, 16, 302–312. https://doi.org/10.1016/j.omtn.2019.02.025 Doru Gucer - 12 HRP

Zhuo, Z., Yu, Y., Wang, M., Li, J., Zhang, Z., Liu, J., … Zhang, B. (2017). Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine. International Journal of Molecular Sciences, 18(10), 2142. https://doi.org/10.3390/ijms18102142