Sophie Nguyen's HRP Aptamer Project (2016)

Western blots are vital to myriad fields in scientific research. Their main function is to relatively quantify specific protein levels by comparing the signal it produces to the signal a loading control, such a housekeeping gene (a protein that is produced relatively evenly throughout all cells) like β-actin, produces. Image processing software juxtaposes the signals of the two proteins produced during direct or indirect detection, producing a relative quantification. The results of Westerns have a variety of uses such as characterization of gene mutations or diagnosis of disorders that affect protein expression.

Production of signals, or detection, is vital to Western blots and also drives its cost due to the use of antibodies. Currently, it is common to reuse antibodies multiple times after the nitrocellulose membranes incubate in them; however, this technique often risks the fidelity of the results of the assay due to several factors. One major factor is microbial contamination; enzyme-conjugated antibodies are often diluted in 5% non-fat milk in order to decrease non-specific antibody binding to the membrane but is stored at 4ºC to prevent reduction of enzyme activity. Microbial growth, as well as spoilt milk, is a legitimate concern because of this. In order to negate bloom, many have chosen to include sodium azide, a toxic salt, in their antibody solutions; however, sodium azide is also capable of inactivating horseradish peroxidase, the enzyme essential to visualization. Another factor is reduced binding affinity; many antibodies will not properly adhere to their specific antigens after a period of time as little as one week (Biocompare 2013, Boonrod 2009). The aforementioned horseradish peroxidase (HRP) is a key element in indirect detection. The enzyme is found endogenously in the roots of Armoracia rusticana, or the horseradish plant, and is widely studied due to its ability to amplify weak signals (Adams 1992). It is a monomeric protein

consisting of 308 amino acids, weighs 44 kDa, and is currently utilized in applications such as enzymelinked immunosorbent assay (ELISA), immunohistochemistry, and has recently been studied in neurological applications such as marking neurons (Vietch 2004). An aptamer solution to the costly use of antibodies in Westerns can be found by targeting HRP. Aptamers are oligonucleotides that bind with high affinity and specificity to a chosen target; multiple rounds of systemic evolution of ligands by exponential enrichment (SELEX) will be conducted to select for an aptamer that binds to HRP. There has been success in the past in replacing alkalinephosphatase-linked antibodies with aptamers in ELISA, thus there is potential for aptamers to replace those used in Western blots (Huynh 2015). So far, there has been limited success in creating aptamers targeting HRP (Mir 2006). While this project focuses on providing an alternative for antibodies in Westerns, theoretically any aptamer that is found that binds to HRP can be used for a plethora of different applications, provided they are in vitro per the selection conditions.