Sujana Nelakanti's HRP Aptamer Project (2017)

Development of an HRP-Thrombin Aptamer Complex for Diagnostic Application of Thrombin Related Diseases

Introduction and Background

Over two million people a year are diagnosed, six hundred thousand people are hospitalized every year, and a hundred thousand people suffer fatalities every year from thrombin related diseases (Grosse, 2012). Specifically, a common type of thrombin-related disease is deep vein thrombosis, which is a blood clot that forms in the deep vein. Deep vein thrombosis can lead to another disease called pulmonary embolism, which is when the blood clot from the vein travels to the lung. Together, deep vein thrombosis and a pulmonary embolism, result in a venous thromboembolism, which is the formation of blood clots in the vein (Know VTE).

Coagulation is the process in which liquid blood transforms into a semi-solid state of blood (MedicineNet, 2016). Optimal coagulation requires effective inhibition of thrombin and its effect on platelets (Tran, 2011). Thrombin plays a critical role in coagulation and platelet activation. It is an enzyme that converts fibrinogen into fibrin, this transformation allows for the clotting of blood (Coughlin, 2015).

Currently, there is a lack of focus on the prevention of thrombin-related diseases. Current research is focused on alleviating the symptoms once they already have occurred instead of preventing the problem before it occurs. The target Horseradish Peroxidase (HRP) can be used for the diagnostic purposes, including the labeling of specific proteins for the detection of disease. Many labs use HRP for labeling, specifically, it is used for the detection of proteins using the catalyzation of hydrogen peroxide. Specifically, an aptamer that would selected against the target HRP, would have the potential to serve a diagnostic purpose by detecting amperometric levels of thrombin concentration.

Aptamers are nucleic acid species which have a binding affinity for different targets. These targets can be metal ions, small organic compounds, biological cofactors, metabolites, proteins, and whole organisms (such as bacteria, viruses, yeast, and mammalian cells) (Hicke, 2000). Aptamers are used for medical therapeutics by having inhibitory effects to alleviate the disease, used as diagnostics to detect diseases, and serve as gene regulators or as vehicles for drug delivery (McCauley, 2003). The Sullenger Lab developed a thrombin aptamer with two exosites. The first exosites binds to fibrinogen and the second one mediates the binding of heparin and causes the initial platelet adhesion. When both the exosites are blocked, by using DNA and RNA aptamers that are respective the specific exosites, it results in inhibiting thrombin concentration, which then results in inhibiting platelet concentration (Nimjee, 2009).

Therefore, the goal is to develop a HRP-Thrombin Aptamer complex for detection of high levels of Thrombin Concentration. ELISA (enzyme-linked immunosorbent assay) is a technique used to detect a variety of species such as proteins, peptides, cells, ligands, or even hormones with high sensitivity. On the other hand, a Sandwich ELISA is a technique which simultaneously uses two aptamers in order to capture the target and to report the target detection (Thermo Fisher Scientific). In this complex, the HRP will catalyze the relay-mediation reduction of hydrogen peroxide and the resulting amperometric response will be measured. As a result, the levels of thrombin concentration will be measured.

An RNA aptamer selection for the target HRP is underway with the N40B pool, and currently, the third round of selection is 50% complete. Eventually it will allow for the development of a binding assay that can address the larger of problem developing an HRP-Thrombin Aptamer complex for the prevention of thrombin-related diseases.

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References

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