Kennedy Tham's HRP Aptamer Project (2019)

RNA Aptamer against Horseradish Peroxidase for an Accessible and Low-Cost ELONA Diagnostic of Nipah Virus

Introduction and Background:

With more than 250 cases of febrile encephalitis observed in farm and abattoir workers, the outbreak of the Nipah virus (NiV) in Malaysia in 1998 caused mass panic. Moreover, the high mortality rate and acute course of infection gave urgency to identifying the virus’ pathogenicity and transmissibility (Banerjee et al., 2019). Although no further cases have been reported in Malaysia since 1998, NiV outbreaks have occurred in other countries in South and Southeast Asia. Since its initial outbreak in Malaysia and in Singapore in 1999, NiV has become a near annual threat to Bangladesh and India with about a 70% mortality rate upon infection (Ang, Lim, & Wang, 2018).

In the Malaysia outbreak, the mode of transmission of NiV to humans was through pigs, while in the Indo-Bangladesh outbreaks, human-to-human transmission and nosocomial infections were observed (Banerjee et al., 2019). Consumption of raw date palm sap contaminated by Pteropus fruit bats, NiV’s reservoir host, was identified as the primary cause of one contracting the virus in Bangladesh and India (Banerjee et al., 2019).

Infection by this agent can have a number of neurological and respiratory manifestations, including encephalitis, aseptic meningitis, psychiatric abnormalities, and acute respiratory distress syndrome. Currently, there are no known effective therapeutics for treating Nipah viral infection, although a number of vaccine candidates have been identified (Ang, Lim, & Wang, 2018). Treatment is mainly limited to supportive care and syndromic management of acute encephalitis syndrome (Banerjee et al., 2019). To combat and prevent this severe infection, however, there needs to be a better way of detecting the virus in developing countries with underfunded scientific infrastructure. The medical community needs to be equipped with readily available and affordable diagnostic tests before the acute disease can be treated. Capable of detecting the virus, an enzyme called horseradish peroxidase (HRP) is currently being used to devise an improved NiV diagnostic test.

One current means of diagnosing NiV is through ELISA testing, which provides a high-throughput and inexpensive method for detecting NiV antigens or IgM and IgG antibodies against NiV (Singh et al., 2019). Nevertheless, difficulties in diagnosis exist as there is a general lack of easily accessible, low-cost NiV diagnostic tests and facilities (Aditi & Shariff, 2019). Moreover, while efficient and inexpensive relative to other current NiV diagnostic methods, antibodies for ELISAs still take several months to develop, often show poor specificity, and are traditionally produced in vivo, which may hinder accessibility (Groff, Brown, & Clippinger, 2015). A promising alternative to using antibodies in ELISAs is the use of aptamers. An aptamer is a unique oligonucleotide sequence that can selectively bind to NiV. The benefits of aptamers over conventional antibodies include their affordability, ease of production under in vitro methods, thermal stability, unique and adaptive structures, high specificity, and readability (Lakhin, 2013). These advantages make aptamers useful in a potential NiV ELONA that would be more simple, reliable, accessible, and cost-effective than current diagnostic methods.

An aptamer will be isolated from a nucleic acid library through a process called the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) as shown in Figure 1. SELEX is an in vitro, iterative selection process that attempts to identify an aptamer with the highest affinity to a specific target of interest upon multiple rounds (Kong & Byun, 2013). For the development of an NiV diagnostic tool, the target of interest is HRP, which will ultimately function as a reporter molecule. A pool of RNA sequences is exposed to immobilized HRPs on streptavidin magnetic beads. Through bead-based selection, unbound RNA species are washed away while bound RNA species are eluted and later amplified and purified to generate an enriched pool for further rounds of selection. In other words, SELEX narrows the large pool of random RNA sequences down to a single aptamer.

The target of interest that will function as a reporter molecule for the diagnostic of NiV is the enzyme horseradish peroxidase (HRP) as shown in Figure 2. The enzyme, which is naturally found in the roots of the horseradish plant (Amoracia rusticana), binds to hydrogen peroxide to oxidize a variety of substrate molecules (Krainer & Glieder, 2015). HRP is typically used as a reporter molecule in ELISA assays, which utilize antibodies to detect and quantitate compounds or pathogens, by producing a colorimetric response. When a substrate binds to and reduces hydrogen peroxide, the substrate is converted to a colored or fluorescent derivative, allowing for quantification (Held, 2003). An abundance of research has been conducted using HRP as a target in ELISA-like assays, specifically with aptamers. For instance, HRP and an aptamer have been used to detect alpha-methylacyl-CoA racemase (AMACR), an enzyme that is overexpressed in a variety of cancer types (Zeng et al., 2015). Likewise, HRP’s use as a reporter molecule in an ELISA-like assay will be exploited to develop a simple, reliable, and more cost-effective diagnostic tool for NiV that would be more preferable to current diagnostic methods.

The design of the NiV diagnostic test will be based on the indirect-format ELISA for the purpose of signal amplification. Samples of serum will be used for this screening. Once an aptamer against HRP has been identified, it will be extended by adding bases that are complementary to those of an NiV-bound aptamer, forming an aptamer complex. Once HRP preferentially adheres to NiV through the mediated aptamer complex, the substrate for HRP, TMB (3,3’,5,5’-tetramethylbenzidine), will bind to HRP and will quickly develop a blue color, allowing for simple detection and quantification of NiV via UV-Vis spectroscopy. See Figure 3 for a simplified model of this application.

In the development of this diagnostic application, the first round of SELEX is underway and almost complete. Purifying the bound RNA is in progress. If the purified RNA is recovered, the remaining steps of the SELEX process may proceed in hopes of narrowing the N71 RNA pool and isolating an aptamer that can be used in a diagnostic test for NiV. Such a diagnostic test will be highly specific to NiV, more cost-effective than ELISA tests, and vastly accessible to those battling with NiV infection in developing countries.

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References:

Aditi, & Shariff, M. (2019). Nipah virus infection: A review. Epidemiology and Infection Journal, 147, e95. doi: 10.1017/S0950268819000086

Ang, B. S. P., Lim T. C. C., & Wang L. (2018). Nipah Virus Infection. Journal of Clinical Microbiology, 56(6). doi: 10.1128/JCM.01875-17 Banerjee, S., Gupta, N., Kodan, P., Mittal, A., Ray, Y., Nischal, N., Soneja, M., Biswas, A., & Wig, N. (2019). Nipah virus disease: A rare and intractable disease. Intractable and Rare Diseases Research Journal, 8(1), 1-8. doi: 10.5582/irdr.2018.01130

Groff, K., Brown J., Clippinger A. J. (December 2015). Modern affinity reagents: Recombinant antibodies and aptamers. Biotechnology Advances Journal, 33(8), 1787-1798. doi: 10.1016/j.biotechadv.2015.10.004

Held, P. (2003, April 21). Horseradish Peroxidase (HRP) Determination Using Amplex Red and Synergy HT Multi-Mode Microplate Reader. Retrieved April 8, 2019, from https://www.biotek.com/resources/application-notes/horseradish-peroxidase-hrp-determination-using-ampl ex-red-and-synergy-ht-multi-mode-microplate-reader/

Kong, H. Y., & Byun, J. (2013, November). Nucleic Acid Aptamers: New Methods for Selection, Stabilization, and Application in Biomedical Science. Biomolecules & Therapeutics Journal, 21(6), 423-434. doi: 10.4062/biomolther.2013.085

Krainer, F. W., & Glieder, A. (2015). An updated view on horseradish peroxidases: recombinant production and biotechnological applications. Applied Microbiology and Biotechnology Journal, 99(4), 1611-1625. doi: 10.1007/s00253-014-6346-7

Lakhin, A. V., Tarantul, V. Z., and Gening L. V. (2013). Aptamers: Problems, Solutions, and Prospects. Acta Naturae Journal, 5(4), 34-43.

Singh, R. K., Dhama, K., Chakraborty, S., Tiwari, R., Natesan, S., Khandia, R., Munjal, A., Vora, K. S., Latheef, S. K., Karthik, K., Malik, Y. S., Singh, R., Chaicumpa, W., Mourya, D. T. (2019, April 22). Veterinary Quarterly Journal, 39(1), 26-55.doi:10.1080/01652176.2019.1580827

Zeng, Y., Zheng, A., Wu, J., Cai, Z., Huang, A., & Liu, X. (2015, October 29). Horseradish peroxidase and aptamer dual-functionalized nanoprobe for the amplification detection of alpha-methylacyl-CoA racemase. Analytica Chimica Acta Journal, 899, 100-105. doi: 10.1016/j.aca.2015.10.007