Alkaline Phosphatase (AP) is implicated in numerous disease states (such as Paget's disease), as well as an important physiological marker. Additionally, it is a ubiquitous reporter used in molecular biology labs around the world.  In collaboration with Eric Wei, we sought to develop a multifaceted tool that may serve both diagnostic purposes and molecular switch/reporter applications. To this end, we isolated and identified such a tool in the form of an aptamer (nucleic acid binding species) against Calf Intestinal AP (CIAP).Anti-CIAP aptamers were isolated using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX).  Beginning with a diversified pool of RNA (�R50� pool), iterative rounds of selection were performed to wash away the non-binding RNA species and enrich for RNA species specifically bound to CIAP. After nine rounds of selection, a reoccurring 14-mer RNA motif (named VDH2.14 and identified in 15 of 40 clones sequenced), presented itself in a similar loop structure for almost all of the RNA folded structures.  Furthermore, radioactive binding assay results revealed that the RNA clones containing the 14-mer motif had significantly greater binding to CIAP (approximately 60% more binding) than RNA clones without the motif.The identification of anti-CIAP aptamers has potentiated the development of tools useful for diagnostic and reporter/switch applications.  While currently untested, the human ortholog could serve as a target for the CIAP, which shares 70% amino acid sequence identity.  Additionally, preliminary results indicate the anti-CIAP aptamers may have an inhibitory effect on CIAP. This work will focus on the isolation, identification, and characterization of the anti-CIAP aptamers, with a diagnostic applications approach, while the kinetic characterization of the aptamers and the molecular reporter application will be reserved for Eric Wei's discussion.

Is it a ligand switch or a dipstick diagnostic tool? Yes! RNA aptamer against calf intestinal alkaline phosphatase

247th National Meeting (2014)

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