Johanna Lindquist's CIAP Aptamer Project (2018)

Nucleic Acid Aptamer Against Calf Intestinal Alkaline Phosphatase for The Detection of Coronary Artery Disease

Cardiovascular disease takes 17.7 million lives each year, making it the fatal disease in the world (World Health Organization [WHO], 2018). In the United States, coronary artery disease (CAD) is the most common type of heart disease (Harvard Health Publishing, 2018). Coronary Artery Disease is considered a “silent killer” because there are often no symptoms of the underlying disease until the event of a heart attack or stroke. Despite the prevalence of the disease, a test for CAD is not part of a routine check-up and is inaccurate and expensive. As over three quarters of CAD deaths are in middle- and low-income countries, the use of aptamers could provide an affordable way to detect and monitor cardiovascular disease in patients worldwide (World Health Organization [WHO], 2018). High levels of low-density lipoprotein (LDL) cholesterol is a precursor to CAD and, until recently, has been the primary focus of physicians in identifying a patient’s risk for the disease. However, a 2005 Harvard Women’s Health Study has identified the C-reactive protein (CRP) as a more accurate predictor of CAD than LDL cholesterol (Harvard Health Publishing, 2018). CRP is a protein that is produced by the liver in reaction to inflammation and contributes to atherosclerosis as it stimulates inflammation and promotes clotting (Orito, et al., 2012). Coronary inflammation is the body’s response to fatty buildup in the coronary arteries which results in plaque rupturing and serious, fatal heart attacks (World Health Organization [WHO], 2018). Since the inflammation is focused in such a small area of the body, it only produces a slight elevation of CRP levels and remains within the “normal range” (Harvard Health Publishing, 2018). A simple blood test is unable to detect this change as it measures changes in mg/L whereas aptamers can target molecules as small as 60 kDa and they are 12-30 kDa in size allowing for them to detect very slight changes in CRP levels (Orito, et al., 2012). By implementing aptamers in a blood sample, the cost of detection tools will decrease, and the identification of discrete inflammation will be possible. An aptamer against the C-reactive protein would display a high affinity and specificity allowing for precise testing of CAD, and due to the cheap and easy production of aptamers, this diagnostic tool would be more readily available to patients than the current blood test (Lakhin, Tarantul, & Gening, 2013).

An aptamer is an oligonucleotide that has a high-affinity and specificity to bind to a target molecule and enact beneficial processes such as therapeutics, drug delivery, or diagnostics (Figure 3). The single-stranded RNA or DNA oligonucleotides are non-immunogenic and non-toxic; and when compared to their nucleotide analogues, antibodies, aptamers are more cost-effective and have high affinities and specificities for a wide range of targets (Ruscito & DeRosa, 2016).

An Enzyme-Linked Oligonucleotide Assay consisting of an aptamer against the C-reactive protein and an aptamer against Calf Intestinal Alkaline Phosphatase (CIAP) can be utilized as a diagnostic of coronary artery disease (Figure 4). CIAP is an enzyme found in bovine intestine that catalyzes the hydrolysis of 5’ phosphate groups from nucleic acids and nucleotide triphosphates to prevent re-ligation of DNA and labeling of the phosphorylated ends (Promega, 2018). In doing so, CIAP maintains intestinal homeostasis and protection in calves (Lalles, 2010). It is a dimeric enzyme with two equal subunits and a molecular weight of 69 kDa (SIGMA, 2018). Additionally, it has an optimal pH of 9-10 and an isoelectric point of 5.7 (Worthington Biochemical Corporation [WBC], 2018). It is commonly used in bioassay applications, such as an ELONA, because it is an effective reporter molecule that produces a colorimetric response by phosphorylating PNPP (Harvard Health Publishing, 2018). The response is easily detected through fluorimetry. CIAP is currently being researched at the University of Texas at Austin where Vincent Huynh has developed an aptamer against CIAP and connected it to an aptamer against PDGF in an ELONA for the diagnostics of Paget’s disease. Research will be done with the N71 RNA pool in search of an aptamer with a higher affinity for CIAP and an aptamer against the C-reactive protein to construct an ELONA. By quantifying the colorimetric response, CRP levels can be precisely measured to detect coronary artery disease. Aptamers are generated through a selection process known as the Systematic Evolution of Ligands by Exponential Enrichment or SELEX, which consists of selection, partitioning, and amplification (Figure 3). This iterative process is repeated with increasing stringency to select for the RNA species that most tightly bound to the specified target (Lakhin, 2013). Generally, around six to seven rounds are completed to before a final aptamer is developed.This project will serve as a diagnostic tool for coronary artery disease and any other manifestations of atherosclerosis such as stroke and peripheral artery disease. In future works, the aptamer could be useful in the treatment of CAD by identifying the c-reactive protein and implementing therapy in these specific locations (Kambo, 2016). Bead-based selection, ethanol precipitation, reverse transcription, cycle-course PCR, and gel electrophoresis have been completed in the selection process. Due to a lack of bands during ccPCR, efficacy checks will be completed. The first round of aptamer selection is focused on identifying RNA sequences with affinities for Calf Intestinal Alkaline Phosphatase at a 200 pmol CIAP: 200 pmol N71 pool ratio.

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