Njambi Kimani's HRP Aptamer Project (2018)

Diagnostic Application for Leukemia Utilizing an Aptamer Against Horseradish Peroxidase

Introduction and Background:

Cancer is caused by the unregulated growth of cells that can eventually spread to other places in the body (National Institute of Health, 2007). While there is no cure for cancer, the earlier the cancer cells are detected the higher chance that the patient will survive. If doctors can remove the primary source of cancer and tumors, then the cancer cells will not have an opportunity to metastasize, or spread throughout the body, which becomes even harder for physicians to treat. Since there is no known cure for cancer the only treatment that doctors can provide is chemotherapy, radiation, and surgery (National Cancer Institute, 2017). For this specific research project, leukemia was chosen as the cancer because of its biomarker, miRNA. Leukemia is a specific type of cancer that develops within the blood cells but there are different types including acute myeloid, acute lymphocytic, chronic myeloid, and chronic lymphocytic. (American Cancer Society, 2018) The difference between the types depends on how fast the cancer grows and where it occurs. For acute myeloid leukemia, the overproduction of miRNAs has been detected as a biomarker for this type of cancer (Wang, 2014). Acute myeloid leukemia, AML, is a type of leukemia that is present in the bone marrow that develops rapidly in the cells (Camos, 2006). A diagnostic aptamer can help diagnose patients that may have elevated levels of miRNAs and the colorimetric properties of the target, HRP, will be utilized.

The target of interest is Horseradish Peroxidase, also referred to as HRP. Since HRP is a reporter molecule, the target fluoresces when it is introduced to its substrate, specifically for this application Tetramethylbenzidine, or TMB (Stovall, 2018). A biomarker, which is a biological molecule, can be a tagging system for researchers to detect a disease (Strimbu 2010). In cancer, there is often a mutation in the places that are supposed to regulate cell division and growth. In AML, the proposed biomarker is MicroRNAs or miRNAs. When overexpressed in the cells, it can be a sign of unregulated cell growth which produces the tumors. A toe-hold displacement will be utilized as the detection mechanism. To utilize the miRNA biomarker, an aptamer will be selected against HRP and it will contain the complementary strand to the miRNA sequence. The complementary strand of the miRNA and HRP will be attached to the aptamer. TMB can bind in the presence of HRP once the miRNA biomarker is detected and HRP can produce a fluorescent signal. This application is important because there is a need to detect cancer, specifically acute myeloid leukemia earlier so that patients have a higher chance of treatment and survival.

There are eight main steps in the SELEX process that are repeated to discover the aptamer with the highest binding affinity to the target, shown also in figure 3 (Stovall, 2018). These steps include a pool binding reaction, successive washes, reverse transcription, polymerase chain reaction (PCR), transcription, polyacrylamide gel electrophoresis (PAGE), RNA quantification, followed by binding assay and sequencing. Once all of these steps have been successfully completed it symbolizes one round of aptamer selection. These rounds are continuously repeated, about six to eight times, to adjust the stringency and increase the chances of discovering the best aptamer binder.

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References:

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