Natalie Hernandez's HRP Aptamer Project

Horseradish Peroxidase

Introduction & Background

The rising cost of healthcare is hard to ignore considering its impact on the economy and is necessary to comprehend to prepare for the future. Seventeen percent of national health expenditures is due to cardiovascular disease alone, which is the leading cause of death in the United States (3). By 2030, 40.5% of the U.S. population is projected to have a type of heart disease and direct costs are expected to triple from while indirect costs due to lost productivity are expected to almost double (3). Although life expectancy has increased along with costs, there is much room for improvement in cardiovascular health while controlling costs.

The root of much of the problem includes factors such as high dietary fat intake, obesity, high blood pressure, and high blood lipid levels. This can build up to coronary heart disease, which is where the issue gets complicated. Improved cholesterol tests and analyzation of levels on an individual basis to the population as a whole is the start to improved solutions not only in prevention of further developing of the disease, but also to costs associated. This improvement can occur in the lab or possibly with home health test kits, raising awareness.

Cholesterol tests, when accurate, can be a great indicator of risk of heart disease. Some can differentiate between good cholesterol (found on high-density lipoprotein) and bad cholesterol (found on low-density lipoprotein) on top of quantifying the total cholesterol (2). Determination of cholesterol concentration is a popular assay in biochemistry (2). Total cholesterol, free cholesterol and esterified cholesterol (bonded to fatty acid), can be measured by coupling the enzymes cholesterol oxidase and cholesterol esterase with peroxidase (2). Cholesterol esterase creates free cholesterol from esterified cholesterol while cholesterol oxidase reacts with steroid alcohol to produce hydrogen peroxide, which can be detected by horseradish .

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peroxidase (2).

Horse-radish peroxidase is an enzyme found in the roots of horseradish plants with the ability to oxidize organic and inorganic molecules. When conjugated to other biomolecules (such as biotin, antibodies, and a substrate) the enzyme can be used to amplify a signal and increase a target’s detectability, acting as a diagnostic tool (1). For example, when exposed to “near-ultraviolet light” it is brightly colored, making the enzyme useful in making transparent proteins visible under certain wavelengths (1). Furthermore, horseradish peroxidase readily combines with hydrogen peroxide and together the two molecules can oxidize chromogenic hydrogen donors (4). Horseradish peroxidase, as seen in Figure 1, is a three dimensional glycoprotein with an isoelectric point of 6.0 and a molecular weight of 44,000 Daltons; 33,890 Daltons come from a single polypeptide chain with four disulfide bridges and 9400 Daltons come from carbohydrate content (4) (5). It’s glycosylation, attachment of carbohydrate to functional groups, leads to less non-specific binding (useful in binding nucleic acids) (4).

Because of its qualities, horseradish peroxidase can be used for the detection of cholesterol levels. In order for that to work, it must bind to an antibody. However, using an aptamer instead might make a better, more specific, and more efficient use of horseradish peroxidase. High-binding affinity aptamers (chemically synthesized oligonucleotides, Fig. 2) are more recently being favored over antibodies in the scientific community due to their ability to fold into complex three-dimensional shapes for interaction while distinguishing between similar targets and the target itself by a single amino acid or functional group (6). Aptamers can be selected from a random pool of nucleotide sequences and can be used once they have binded to a target. They are preferred because they are easier and less costly to manufacture, are highly stable, and have the capability of binding to many different types of protein (6).

In order to find an aptamer for a specific target, the process of selection must take place. As seen in Fig. 2, selection begins with a random pool of DNA or RNA being immobilized on desired target on magnetic beads. Weakly bound pool would be washed away and the tightest binders would be eluted and amplified and depending on whether the pool was from RNA, reverse transcription and transcription would occur (6).

In finding an aptamer for horseradish peroxidase and cholesterol, an assay such as the Enzyme Linked Immunosorbent Assay (ELISA) must take place for the application to work. In applying the principle of conjugation mentioned earlier, an indirect ELISA would take place in which an antibody would be attached to the substrate that would be cholesterol (shown as antigen on Figure 3). An aptamer would replace a secondary antibody conjugated to the primary antibody and horseradish peroxidase. Horseradish peroxidase would then hydrolyze in order to recognize the substrate (7).

Successful aptamers have already been found. An application of an aptamer for the detection of thrombin by signal amplification of horseradish peroxidase, for example, has been discovered (9). This aptamer allows for a quick, selective, and inexpensive quantification of thrombin, an enzyme which promotes blood clotting. This shows the potential in protein detection and disease diagnostics in aptamers (9). Currently in the Ellington Lab at the University of Texas at Austin, an RNA aptamer selection for the binding of horseradish peroxidase-biotin for the use of cholesterol level diagnostics is underway. Hopefully, the affinity towards functional groups found in the carbohydrate component of horseradish peroxidase will cause a successful binding of an RNA aptamer. Once conjugated to the successfully binding aptamer, it is expected that horseradish peroxidase will react with hydrogen peroxidase produced by cholesterol enzymes to produce a color change and indicate the presence of lipoproteins carrying cholesterol in the blood, quantifying cholesterol levels. If this happens, we would have found an effective and efficient solution to healthcare availability, cost, and would've found a better alternative to antibodies for HRP.

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Citiations

(1)“Anti-HRP Antibodies." Anti-HRP Antibodies. N.p., n.d. Web. 8 Apr. 2015. <http://www.pierce- antibodies.com/targets/t/Anti-HRPAntibodies.cfm>.

(2) Azevedo, Ana M. "Horseradish Peroxidase: A Valuable Tool in Biotechnology." Biotechnology Annual Review 9.1387-2656 (n.d.): 199-247. Web. 8 Apr. 2015.

(3) Centers for Disease Control and Prevention. “Heart Disease in the United States”. Web. 27 Oct. 2015.

<http://www.cdc.gov/heartdisease/facts.htm>

(4) “Enzyme Explorer - Peroxidase Enzymes." Sigma-Aldrich. N.p., n.d. Web. 8 Apr. 2015. <http:/ www.sig maaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/peroxidase-enzymes.html>.

(5) "Protein Data Bank." N.p., n.d. Web. <.http://www.rcsb.org/pdb/explore.do?structureId=1GWO>.

(6) “Recent Progress in Nucleic Acid Aptamer-Based Biosensors and Bioassays." MDPI. N.p., n.d. Web. 9 Apr. 2015. <http://www.mdpi.com/1424-8220/8/11/7050/htm>

(7) Rockland Antibodies & Assays. “Custom Antibody Conjugation”. Web. 28 Oct. 2015.<http://www.rockland-inc.com/custom-antibody-conjugation.aspx>

(8) Thermo Fisher Scientific. Pierce Horseradish Peroxidase. https://www.thermofisher.com/order/cata log/product/29139?ICID=search- product

(9) Zhao, Jie, Meiling Liu, Youyu Zhang, Haitao Li, Yuehe Lin, and Shouzhuo Yao. "Apoferritin Protein Nanoparticles Dually Labeled with Aptamer and Horseradish Peroxidase as a Sensing Probe for Thrombin Detection." Apoferritin Protein Nanoparticles Dually Labeled with Aptamer and Horseradish Peroxidase as a Sensing Probe for Thrombin Detection. N.p., n.d. Web. 9 Apr. 2015. <http://www.sciencedirect.com/science/article/pii/S0003267012015632>