Justin Yun's HRP Aptamer Project (2016)

Aptamer Selection Against Horseradish Peroxidase for Diagnosing HIV-1

Introduction/Background

The spread of HIV/AIDS epidemic has been growing rapidly across the world, but even more so in poverty stricken regions in Sub-Saharan Africa and in Latin America. In fact, more than 14,000 people are diagnosed with HIV every day in Sub-Saharan Africa, a region where 60% of the population live with less that $1 a day (Mbirimtengerenji, 2007). There is a desperate need for more affordable avenues for HIV diagnosis and treatment as increased affordability would increase accessibility for diagnosis. Creating a more financially attainable diagnosis option for HIV could make for earlier detection and more options when it comes to treatment (Girardi et al. 2007).

A common method of detection for the HIV is through enzyme-linked immunosorbant assay (ELISA) using horseradish peroxidase (HRP) and antibodies. ELISA uses horseradish peroxidase-conjugated antibodies as a reporter enzyme to signal the presence of HIV antibodies in the body. Then a substrate would be added to activate a color change that would determine the presence of HIV. In figure 3, a ELISA is shown using antibody linked enzyme.

The target, horseradish peroxidase, is an enzyme found in the roots of the common Horseradish plant (Armoracia rusticana). When exposed in the proper substrate, HRP can cause a color change which is useful in detecting small amounts proteins, antibodies, and other molecules, making it ideal for use in ELISAs. As seen in figure 1, HRP is about 44kDa and is a monomeric protein that is also used other applications, such as a cytotoxic drug to target cancer cells (Kim et al, 2006).

Aptamers are oligonucleotides that bind to a target molecule with high affinity and specificity. In this case, the aptamer will be an RNA sequence that binds to the protein HRP. Finding an aptamer to replace the antibody conjugated to HRP would be beneficial for many reasons. Aptamer production is cheaper due to its systematic procedure and affordable material and they are also much more resilient to external conditions such as time and temperature, therefore reducing transportation costs significantly (Jayasena, 1999).

Aptamers have a wide variety of uses for diagnostics, therapeutics, and drug delivery. For example, some aptamers have been used as diagnostic tools by binding to biomarker proteins in cancer cells. An aptamer found for HRP would be used as a diagnostic tool for HIV detection. Theoretically, HRP could be used to diagnose diseases other than HIV. Notably, since the main application of HRP and its corresponding aptamer can be used in ELISA, other diseases that are normally detected with antibody mediated ELISAs can be detected using aptamers conjugated to HRP. HRP is proven to be stable in PBS buffer (Alzevedo et al, 2003), but there seem to be no unique structures or RNA binding domains that would be useful for nucleic acid binding.

Currently, there are no aptamers found for the protein HRP. However, the selection process for an aptamer is currently at round 2 of selection where the RNA pool will be exposed to the target and unbound will be washed. Many more rounds must be completed before going onto the next steps to identify the aptamer. If this aptamer is found, the development of a more affordable diagnostic tool for HIV detection could come into fruition.

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References