Therapuetic RNA aptamers against Calf Intestinal Alkaline Phosphatase

Vincent Huynh

Abstract:

Gram-negative bacteria have a lipopoylsaccharide layer which is notable for inducing inflammatory responses in humans. Some examples of pathogenic gram-negative bacteria are E. coli, Salmonella, Shigella, and Legionella. One proposed mechanism for the survival of bacteria in phosphate-deprived environments is the role of alkaline phosphatase. Researchers at the National Institute of Health in Osaki Tokyo have observed that bacteria grown in media with low phosphate levels have increased formation of alkaline phosphatase [1]. A RNA ligand that can bind to bacterial alkaline phosphatase can inhibit its function to produce phosphates and therefore restrain bacterial growth by phosphate starvation. Although this research focuses on calf intestinal alkaline phosphatase (CIAP), the results are very applicable to bacterial alkaline phosphatase. Researcher Jennifer Murhey et al at the Boston College Department of Chemistry have found that bacterial and mammalian alkaline phosphatases have 25-30% conserved sequences [2]. Of this percentage, they have found that the active sites of the bacterial and mammalian alkaline phosphatases are completely identical with the exception of three amino acids [2].

Specific Aim #1: The first aim of this research is to find an aptamer against the calf intestinal alkaline phosphatase through a bead-based method called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Current research has shown that an aptamer against CIAP is highly probable. Six rounds of selection were performed and the products of the selection were then quantitated using a bead-based binding assay. The binding assay was performed in the Ellington Lab which uses radioactive ATP to tag the nucleic acid species. The results of the binding assay supported that there was an increased binding for CIAP from 7.60% in round 1 to 11.69% in round 6. Furthermore, background binding decreased from 6.03% in round 1 to 2.06% in round 6. These results suggest that selection is working and that there is an aptamer for calf intestinal alkaline phosphatase.

Specific Aim #2: The second aim of this research is to test the specificity of the CIAP aptamer to bacterial alkaline phosphatase. Selection or modification of the CIAP aptamer can be performed to accommodate the minor differences between the two alkaline phosphatases. Once an aptamer against bacterial alkaline phosphatase is derived, the therapeutic use of the aptamer should be researched by testing the inhibitory effects of the aptamer on bacterial pathogenesis by phosphate starvation.

1mg of biotinylated alkaline phosphatase at a concentration of 1.1mg/ml can be purchased from Thermo Scientific for $147. It should cost around $3.68 per selection (assuming 400pmol per selection). The molecular weight of CIAP is 69 kDa. The product number of biotinylated alkaline phosphatase is 29339. Thermo Scientific can be contacted at 815-968-0747.

Below is an outline of the project

 

References

[1]   Horiuchi, T., S. Horiuchi, and D. Mizuno. "A Possible Negative Feedback Phenomenon Controlling Formation of Alkaline Phosphomonoesterase in Escherichia Coli." Nature.com. Nature Publishing Group, 30 May 1959. Web. 18 Sept. 2012. <http://www.nature.com/nature/journal/v183/n4674/abs/1831529b0.html>.

[2]   Murphy, Jennifer E., Thomas T. Tibbitts, and Evan R. Kantrowitz. "Mutations at Positions 153 and 328 In Escherichia ColiAlkaline Phosphatase Provide Insight Towards the Structure and Function of Mammalian and Yeast Alkaline Phosphatases." The Crystal Structures of Three Mutant Alkaline Phosphatases (1995): n. pag. Science Direct. 1995. Web. <http://ac.els-cdn.com/S0022283685705761/1-s2.0-S0022283685705761-main.pdf?_tid=c2a7e91e-01bc-11e2-bd56-00000aab0f02&acdnat=1347992280_82d9adf77275fe97aa8d43aa83f0f832>.

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