Hannah Kambo's CIAP Aptamer Project (2016)

Colorectal cancer, or colon cancer is a deadly disease which is increasingly affecting younger individuals [1]. Tumor markers are produced in these and other cancers, as well as benign conditions, but are present in much more elevated levels in cancerous conditions [7]. A commonly used tumor marker in diagnostic tests is carcinoembryonic antigen (CEA), which is often used as an indicator of cancers of the colon and rectum [8]. Alkaline phosphatases are another type of tumor marker frequently used in detecting cancers within the gastrointestinal tract. These enzymes are produced in various parts of the body in response to conditions such as Paget’s disease and other disorders of the bone, gastrointestinal afflictions, and some forms of cancer [9]. In a study from the European Journal of Cancer, these enzymes were found to be more reliable indicators of a malignant tumor than CEA [10]. Alkaline phosphatases are found in both prokaryotes, such as Escherichia coli, and eukaryotes, such as cows and humans [11] (Figure 2). These enzymes are located in a number of areas of the mammalian body. Alkaline phosphatases are particularly concentrated in the intestines and placental region, but are also found in the liver, kidney, brain and bones [12].

The current research involves calf intestinal alkaline phosphatase (CIAP), which is extracted from the intestinal mucosa of cows [13]. CIAP has a molecular weight of 69,000 Da (69kDa), an optimal pH of 9-10, and an isoelectric point of 5.7 [14]. Alkaline phosphatases exist in a dimeric quaternary structure with two identical subunits. The active site contains two zinc ions and one magnesium ion [13][15][16]. Bead-based aptamer selection will be utilized in this investigation. This involves an in vitro SELEX method of immobilizing a target with beads then introducing an RNA pool to the target (Figure 3). Previous investigations involving CIAP have focused on diagnostics and therapeutics for illnesses such as Paget’s disease and Crohn’s disease. This research has been performed within the Aptamer stream in the Freshman Research Initiative, which is an extension of the work within the Ellington lab [17].

Aptamers are sequences of DNA or RNA which bind to a target molecule strongly and with high specificity. Aptamers are valued over antibodies for their stability, ease of modification, and lack of negative immune effects [18]. These factors make aptamers a desirable choice in the development of a more reliable detection tool for elevated ALP levels in colorectal cancer patients. The current method of testing for alkaline phosphatases is through a serum, urine or blood test for these enzymes, called an ALP test [19]. While cost-effective, concerns have been raised about the generation of false positives or negatives through these tests, and those with greater specificity and sensitivity are being sought [10]. Aptamers can be used in place of antibodies within a sandwich enzyme-linked immunosorbent assay (ELISA) with a colorimetric response [20]. This is useful in not only diagnostic applications, but also for the purpose of prognoses. Tracking the levels of alkaline phosphatases over time can be a good indicator of the ability of colorectal cancer to relapse or metastasize.

Bead-based aptamer selection will be utilized in this investigation. This involves an in vitro SELEX method of immobilizing the CIAP target on biotinylated streptavidin beads then introducing an RNA pool to the target. CIAP comes in a buffer solution of pH 8.0 (10 mM Tris, 0.15 M NaCl, 0.1 mM ZnCl2, 0.1 mM MgCl2) in 50% glycerol [14] A previous aptamer selection was conducted to develop a biosensor which disinhibited CIAP after exposure to a particular target [ 21].

In the current selection, the first round been completed, and the second round is in progress. Immobilization of the CIAP target to the beads and RNA incubation have been performed. Precipitation and reverse transcription to obtain the single-stranded DNA for cycle-course PCR has been completed. Amplification of the DNA product through cycle-course PCR and large-scale PCR have also been performed. Reverse transcription and cycle-course PCR will be repeated. Aptamer selection against alkaline phosphatases is a first step toward developing a novel colorectal cancer surveillance tool. This will ensure that colorectal cancer patients have a means of adequately tracking disease progress, thereby improving chances of recovery.

[1] Colon Cancer Alliance (2016). What Is Colon Cancer.

[2] Mayo Clinic (2016). Test ID: ALP.

[3] Cancer Treatment Centers of America (2015). Tumor Markers.

[4] Oncology Pro (2016). P-120-Serum alkaline phosphatase as an early diagnostic tool in colorectal cancer.

[5]Wei, JS; Chung, NC; Wei, LL; Tzeng, WF; Liu TZ; Wang JY (1993). High-molecular-mass alkaline phosphatase as a tumor marker for colorectal cancer: comparison of two test methods. Clinical Chem,39(3):540-3.

[6]Keefe, Anthony D; Pai, Supriya; Ellington, Andrew (2010). Advantages and limitations of aptamers versus antibodies. Nature Reviews Drug Discovery. 9, 537-550.

[7] Tumor National Cancer Institute (2015). Tumor Markers.

[8] Cancer Treatment Centers of America. Web (2015). Tumor Markers.

[9] Mayo Clinic (2016). Test ID: ALP.

[10] Aabo K, Pedersen H, Kjaer M.(1986). Carcinoembryonic antigen (CEA) and alkaline phosphatase.

Eur J Cancer Clin Oncol. 22(2):211-7.

[11] Zhang L, Buchet R, Azzar G .(2004). Phosphate Binding in the Active Site. Biophys J.86(6): 3873–3881.

[12] Lindon G, Alais C. (1978). Alkaline phosphatase in human, cow and sheep milks: molecular and catalytic properties and metal ion action.

[13] Invitrogen Corporation (2003). Calf Intestinal Alkaline Phosphatase(CIAP).

[14] Affymetrix (n.d.) Calf Intestinal Alkaline Phosphatase (CIAP, CIP).

[15] Boulanger, Jr R, Kantrowitz, E. (2003). Characterization of a Monomeric Escherichia coli Alkaline Phosphatase Formed Upon a Single Amino Acid Substitution. The Journal of Biological Chemistry, 278,23497-23501.

[16] Zhifang, C., Zhen X., Yongdoo P, Haimeng, Z. (2001). Activation of Calf Intestinal Phosphatase by Trifluoroethanol. Tsinghua Science and Technology. 5:426-431.

[17] Freshman Research Initiative (2016). Aptamer Stream.

[18] Aptamers(2013). Aptamers vs. Monoclonal Antibodies: A Comparison.

[19] Biocompare (n.d.). Alkaline Phosphatase (ALP) Test Kit from MyBioSource.

[20]BioVision (n.d). Alkaline Phosphatase Activity Colorimetric Assay Kit.

[21]Cabrero, P. (2014). Calf Intestinal Alkaline Phosphatase Aptamer Based Biosensors. McMaster University

[23] Aptamers(2013). Aptamers vs. Monoclonal Antibodies: A Comparison.

[24] UniProt(2016). Alkaline phosphatase, tissue-nonspecific isoenzyme