Vishnu Nair's HRP Aptamer Project (2017)

Combination of Horseradish Peroxidase Aptamer and Nanoparticles for Higher Sensitivity HIV Detection: Final Progress Report

Introduction and Background

In the modern world, one of the most concerning issues would be the increasing number of individuals contracting the Human Immunodeficiency Virus (HIV) leading to possibly fatal consequences. This virus has the capability to cause tremendous stress to the individuals that contract it while possessing high rates of death. This virus is one that severely weakens the immune system and if ignored, can develop into Acquired Immunodeficiency Syndrome (AIDS). This in turn can lead to serious illnesses and even death. Western blot tests and enzyme-linked immunosorbent assay’s are used to detect and measure antibodies in the blood. The types of antibodies found can then be examined to determine what type of disease is present in a patient (Kinman 2015). Since this virus is transferred through sexual contact and body fluids (blood and breast milk), it can be rapidly spread in a population if overlooked. In order to control such a devastating type of ailment, it is of utmost importance that we develop and drastically improve methods to detect precursors for it to deliver treatment as soon as possible. This study will utilize an aptamer for Horseradish Peroxidase to bind the p24 antigen.

Horseradish Peroxidase (HRP) is an enzyme that has the capability to become a diagnostic tool. This agent is found in the horseradish herb, which is cultivated primarily for consumption of its nutritious roots. It weighs 44 kDa and has a three-dimensional structure, having two metal centers comprised of an iron heme group and two calcium atoms (LibreTexts 2013). The peroxidase found in the plant can be used to oxidize various types of compounds. The production of this compound has a plethora of commercial uses including clinical diagnostics and immunoassays (Veitch 2004). Previous work with HRP has produced promising results for cancer therapy. In the Gray Cancer Institute, HRP is first delivered to a tumor followed by the administration of a prodrug. HRP then functions to convert the prodrug into a cytotoxin that will reduce the tumor (Greco, Rossiter, et al. 2001). Due to its high versatility, this study will test the limits of this molecule by focusing it upon the realm of HIV.

For enzymes such as HRP, researchers worldwide have been discovering an increasing number of aptamers for a variety of uses. Aptamers are short oligonucleotide sequences that possess the capability to bind to larger target molecules such as proteins. These molecules have a variety of applications including therapeutics, diagnostics, and signaling. They can bind to target proteins that are precursors to diseases and inhibit them, mitigating the effects of the disease. Aptamers could bind to certain molecules that indicate disease and report the presence of it through fluorescence or other mechanisms. Drug delivery and gene regulation are fields in which aptamers can aid as well. In Li laboratory, aptamers were utilized for the detection of protein biomarkers in the blood to diagnose diseases and begin treatment (Li et al. 2008). The response signal of a surface aptamer-protein-antibody complex was amplified using a reaction that was catalyzed by the horseradish peroxidase enzyme. This became known as the SPRI (surface plasmon resonance imaging) method for detecting thrombin at specific concentrations in human blood (Li et al. 2008).

The aptamer found against the horseradish peroxidase enzyme will be used for diagnostic applications initially. The p24 antigen, a protein that is present in copious amounts in an HIV patient, will be targeted using the created aptamer nanoparticle combo. Once the protein binds to the molecule, HRP will fluoresce, indicating the presence of HIV. Western blot testing and ELISA tests are currently used diagnose HIV. Finding an aptamer for HRP could possibly allow for the sensitivity of these tests to increase, allowing them to sense and detect such antibodies at lower concentrations. Much like Zeng laboratory has done, utilizing an aptamer in tandem with various molecules (nanoparticles) will allow a greater chance for amplified detection (Zeng 2015). Using their aptamer combination, they were able to detect a form of cancer present in two less orders in magnitude (Zeng 2015). This will allow for patients with the virus to be diagnosed earlier and thus treated in the initial stages. An aptamer can be found more easily for this target due to the presence of heme groups that better allow them to attach to proteins. Since the pH level of the PBS buffer is approximately 7.4 and resembles that of human blood, it will be used as a selection buffer to simulate internal body systems and thus find an effective aptamer.

Previous works have led to the discovery use of HRP, aptamers, and nanoparticles simultaneously. Alpha-methylacyl-CoA racemase (AMACR) is a highly expressed protein in various cancer types (Zeng et al. 2015). In the experiment, HRP was immobilized and a thiol-terminated aptamer was used alongside a gold nanoparticle. This allowed for the detection limit of the AMACR protein to be reduced to three orders lower than before due to enzymatic cycling and enzyme loading (Zeng 2015). Since the target protein is detectable at such low amounts, it is possible that certain cancer types can be detected at earlier stages and diagnosed accordingly. Similarly, another aptamer nanoparticle and HRP combination was used for the amplified detection of thrombin in the blood (Zhao et al. 2013). The aptasensor that was created from various affinity reactions allowed for the detection signal for thrombin to be amplified significantly (Zhao et al. 2013). This method, in other words, allowed for thrombin to detected in a more efficient and inexpensive way. Such discoveries continuously increase the impact of aptamers on diagnostics and therapeutics overall. In my project, the aptamer and nanoparticle combo will specifically bind to the p24 antigen, a protein that is present in significant amounts in the body of an HIV patient. Once p24 binds to the molecule, HRP will fluoresce, indicating the presence of HIV.

An RNA aptamer selection is underway, and we hope to observe the true capability of the horseradish peroxidase enzyme in binding specific proteins. With its heme groups, we believe that it has the potential to bind to a wide variety of proteins. We hope to see that this enzyme can have tremendous impact on human lives after running numerous in vitro experiments. Thus far, 50% of the second round of the anti-HRP aptamer selection has been done. Specifically, we aim to find an aptamer against HRP and coat it to chosen nanoparticles in hopes of increasing sensitivity to p24 antigen detection. This is a protein that is present in significant amounts in an HIV patient. Increasing the sensitivity to such proteins will allow for a patient to be diagnosed in far earlier stages, allowing for a faster recovery. When in vitro tests are done with blood serum containing p24 antigen, it is hoped that the protein binds to the aptamer nanoparticle combo. Once bound, HRP will fluoresce, indicating the presence of HIV in the patient. This mechanism is shown in Figure 1.

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