Judy Al-sayyad's CIAP Aptamer Project (2016)

Just from the United States alone, there are approximately 200,000 new cases of Paget’s disease of Bone each year; in fact, as of 2014, over one million individuals suffered from this painful disease (National Institutes of Health Osteoporosis and Related Bone Diseases 2013). Osteitis deformans, also known as Paget’s disease, is a disorder that interferes with the body’s natural recycling process in which new bone tissue gradually replaced the old bone tissue (Ralston 2016). If not quickly identified, the disease can lead to fragile and misshapen bone structure. Because of this, many severe complications can arise which may include: bone cancer, deafness due to the increased compression of the nerves in the inner ear, heart failure, kidney stones, and even paraplegia (partial paralysis) (Ralston 2016). Although the cause of this disease is still unknown, Paget’s disease tends to affect individuals who are older than the age of forty and people who live in certain geographical areas including: the United States, Western Europe, Australia, and New Zealand (National Institutes of Health Osteoporosis and Related Bone Diseases 2013). In addition, individuals who suffer from Paget’s disease are reported to have elevated levels of monoclonal Immunglobin M, or IgM, proteins in their serum (Buxbaum et al. 1983). IgM is an antibody that is produced by B cells in bone marrow and is the first antibody to appear in response to an antigen (The American Heritage Dictionary of the English Language 2004). IgM antibodies are produced when an infection occurs, and if there are increased levels of these proteins this usually will indicate a new infection in the body (Sharma 2007). From this information, the development of Paget’s disease is believed to be linked to the amount of IgM in the body. Ultimately, an aptamer against CIAP could be produced to detect the overexpression of this protein and easily identify whether the patient has Paget’s disease.

An aptamer against Calf-Intestinal Alkaline Phosphatase, more commonly known as CIAP, will be used in this application. For diagnostic purposes, the aptamer will be used in an ELISA. An ELISA is an enzyme-linked immunosorbent assay that determines the presence of a protein within a test sample by producing a color-changing substrate. In this application, an Indirect ELISA will be used to determine the presence of IgM within a patient’s sample. This will be done by replacing one antibody with the aptamer against CIAP and the other with the IgM protein. Colorimetric sensors will also be attached to the aptamer to detect how much IgM is found; the degree of color change will indicate whether the patient has the disease due to increased levels of IgM. Overall, the goal is to detect this disease as early as possible to prevent the arrival of those symptoms mentioned earlier.

Aptamers will be used for this particular experiment. Aptamers are unique oligonucleotide sequences that bind to specific targets with very high affinity and specificity. The role of aptamers is that they function as receptors for target molecules due to their ability to fold into distinct 3D structures. Aptamers are synonymous and are often compared to antibodies because of their similarity in function; however, aptamers possess several key advantages such as: they are easier and more economical to produce, and they are capable of greater specificity and affinity to targets than some antibodies. In addition, they are overall more stable because they can stand repeated rounds of denaturation/renaturation while also tolerating wider ranges of temperature, pH, and salt concentrations all which contribute to a longer shelf life.

The SELEX Bead-Based method is used to produce an aptamer for a specific target. SELEX stands for Systematic Evolution of Ligands by Exponential Enrichment. The process begins by immobilizing the protein target, in this case CIAP, onto the beads and washing away any unbound target. Then, the immobilized target will be mixed with the heat-denatured pool. Next, we elute the RNA which means we will collect the target-bound RNA species. Then, we perform an ethanol precipitation. And lastly, we amplify the pool by doing reverse transcription, cycle course PCR (ccPCR), large scale PCR (lsPCR), transcription, and PAGE (polyacrylamide gel electrophoresis). The SELEX process is repeated until we have reached the desired number of rounds and stringency of the aptamer. Figure 3 shows this process through a visual image.

Calf-Intestinal Alkaline Phosphatase, or CIAP, is a dimer molecule which means it consists of two identical molecules linked together. Figure 4 shows the structure of this molecule. CIAP is also an enzyme that is used to catalyze the dephosphorylation of 5’ and 3’ ends of DNA and RNA phosphomonesters which can be used for subsequent use in cloning since the fragments cannot ligate due to the lack of the 5’ phosphate group (ThermoFisher Scientific 2016). As the name suggests, CIAP is found in the mucous membranes of calf intestines and weighs about 69kDa with an isoelectric point of 5.7 (Worthington Biochemical Corporations 2016). This molecule is also negatively charged which can result in problems binding to DNA and RNA since they are also negatively charged. However, the enzyme can be activated by most cations like Ca2+ and Mg2+(Worthington Biochemical Corporations 2016). Furthermore, CIAP has an optimum pH of 9.8, so it is stable in a buffer with a relatively basic pH value (Worthington Biochemical Corporations). Current research on CIAP is being conducted at the University of Texas at Austin. An aptamer has been developed for this target by UT’s very own, Vincent Huynh. In addition, I am also conducting research on selecting an aptamer against CIAP with UT’s FRI Aptamer Stream. As of now, two rounds of selection have been completed and the third round is currently underway. The goal of this is to complete about 6 rounds of selection to see whether or not an aptamer has been found.

Boghog2. Cartoon Diagram of the Dimeric Structure of Bacterial Alkaline Phosphatase Based On the PDB 1ALK coordinates. Digital Image. 2008.

Buxbaum, J.N, and Sandra Kammerman. Immunoglobin Abnormalities in Paget’s Disease of Bone, 1983, n.p.

Reyes, P.E.C. “Calf Intestinal Alkaline Phosphatase: Aptamer Based Biosensors”. McMaster University: Department of Chemical Engineering. 2014.

"Immunoglobulin M". The American Heritage Dictionary of the English Language (Fourth ed.). Houghton Mifflin Company. 2004.

Netter Images. Illustration of Paget’s Disease of Bone from the Netter Collection. Digital image. 2005.

“Phosphatase, Alkaline.” – Worthington Enzyme Manual. Worthington Biochemical Corporations. 2016.

Ralston, S.H. Paget disease of bone. In: Goldman L, Schafer AI, eds. Goldman's Cecil Medicine. 25th ed. Philadelphia, PA: Elsevier Saunders; 2016, Chapter 247.

Sharma, A. “What do IgG and IgM indicate?”. Doctor NDTV…For the Better Health of Indians. 2007.

ThermoFisher Scientific. CIAP (Calf Intestinal Alkaline Phosphatase). 2016

What is Paget's disease of bone? National Institutes of Health Osteoporosis and Related Bone Diseases: National Resource Center. 2013.