Ruthvik Allala's CIAP Aptamer Project (2015)

Aptamer Against Calf Intestinal Alkaline Phosphatase for Determination of Appropriate Anti-Inflammatory Medication Dosage

Introduction & Background

Adequate levels of gut microbiota, microbe populations living inside the intestine, are essential for a multitude of bodily functions. These functions include digesting certain foods,aiding with the production of vitamins B and K, and maintaining the intestinal mucosa bycombating foreign microorganisms that penetrate the body (Guinane and Cotter, 2013). Adisturbance to the level of the gut microbiota leads to rise in the number of foreignmicroorganisms that results in deleterious effects including digestive inflammation and ulcers(Guinane and Cotter, 2013). As observed in experimental mice, these symptoms can have long-term deleterious effects such as gastritis, an irreversible decay of stomach lining, that shorten the lifespans of these mice (Estaki et al., 2014).

Calf intestinal alkaline phosphatase (CIAP) is an enzyme that aids in the maintenance of the intestinal mucosa by detoxifying foreign microbial ligands (Malo et al., 2010). The molecular weight of the enzyme, a homodimer in nature, is 69 kDa (Alkaline 2015). The general enzyme intestinal alkaline phosphatase is found in a multitude of animals ranging from mice to humans. Research has shown that the usage of calf intestinal alkaline phosphatase, naturally found in intestinal tissue of calves, as a medication can cause a beneficial rise in gut microbiota, which could then register a homeostatic effect by competing foreign microorganisms (Malo et al.,2010). Currently, there are cases utilizing calf intestinal alkaline phosphatase as a tool for an enzyme-linked immunosorbent assay (ELISA) and as medication to maintain the gut microbiota levels of the intestinal tract. The Aptamer Research Lab at the University of Texas at Austin and other labs around the world are currently exploring the health care potential of CIAP.

The rapidly advancing field of biotechnology has brought about a host of useful tools for microbiological research including aptamers, highly specific oligonucleotide binding species. An aptamer binds to selected targets such as proteins with very high affinity. An RNA segment from an RNA pool binds to the target that is bound to the streptavidin beads. This innovative technology can serve as a diagnostic, treatment, or delivery agent. An aptamer that binds to CIAP will be isolated for and then used in a technique known as an ELISA in order to determine the appropriate dosage of CIAP that must be ingested for the body to return to normal gut microbiota levels (Alam et al., 2014). If found, an aptamer against CIAP can be used as a diagnostic tool. However if this protocol is unsuccessful, isolating for an aptamer capable of serving as a treatment for a lack of gut micriobiota will be explored (Yang et al., 2015).

An alternate bead based selection scheme in which the epitope of CIAP will bind to Vincent Huynh’s minimized aptamer variant and allow for a new aptamer from the N71 pool to bind to the active site of CIAP will be used in this selection process. Figure 2 shows the cycle that will be followed in order to complete a single round of selection. By isolating for an aptamer that binds to the active site, a CIAP inhibitor is found. If the minimized variant is not used, an aptamer may also bind to an epitope. This would have the same effect of not registering a significant change in enzymatic activity. Utilizing the minimized variant reduces the risk of Vincent Huynh’s aptamer being amplified during PCR instead of an aptamer isolated from the N71 RNA pool. Multiple rounds of selection lead to a more specified Aptamer.

The RNA aptamer selection is underway and once five rounds are complete, sequencing and binding assay can be performed to determine whether an aptamer has been successfully isolated for. If an aptamer has been isolated for, an ELISA can be then developed in order to facilitate the diagnosis of CIAP levels in cases of inflammation.

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Citations

1. Alam, S.N., Yammine, H., Moaven, O., Ahmed, R., Moss, A.K., Biswas, B., Muhammad, N., Biswas, R., Raychowdhury, A., Kaliannan, K., et al. (2014). Intestinal Alkaline PhosphatasePrevents Antibiotic-Induced Susceptibility to Enteric Pathogens. Ann Surg 259, 715–722.

2. “Alkaline Phosphatase, Calf Intestinal (CIP).” (2015). Product Information. New England Bio

3. Estaki, M., DeCoffe, D., and Gibson, D.L. (2014). Interplay between intestinal alkaline phosphatase, diet, gut microbes and immunity. World J Gastroenterol 20, 15650–15656.

4. Guinane, C.M., and Cotter, P.D. (2013). Role of the gut microbiota in health and chronicgastrointestinal disease: understanding a hidden metabolic organ. Therap Adv Gastroenterol 6,

5. Hedin, C., van der Gast, C.J., Rogers, G.B., Cuthbertson, L., McCartney, S., Stagg, A.J., Lindsay, J.O., and Whelan, K. (2015). Siblings of patients with Crohn’s disease exhibit a biologicallyrelevant dysbiosis in mucosal microbial metacommunities. Gut.

6. Malo, M.S., Alam, S.N., Mostafa, G., Zeller, S.J., Johnson, P.V., Mohammad, N., Chen, K.T.,Moss, A.K., Ramasamy, S., Faruqui, A., et al. (2010). Intestinal alkaline phosphatase preservesthe normal homeostasis of gut microbiota. Gut 59, 1476–1484.

7. Manickum, T., and John, W. (2015). The current preference for the immuno-analytical ELISA method for quantitation of steroid hormones (endocrine disruptor compounds) in wastewater in South Africa. Anal Bioanal Chem.

8. Yang, Y., Millán, J.L., Mecsas, J., and Guillemin, K. (2015). Intestinal Alkaline Phosphatase Deficiency Leads to Lipopolysaccharide Desensitization and Faster Weight Gain. Infect. Immun. 83, 247–258.