Development of a DNA Aptamer that Specifically Inhibits Human Carbonic Anhydrase II

Afroz Karim, University of Texas at El Paso


Carbonic anhydrases (CA) are a family of enzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate. Carbonic anhydrase II is the most abundant protein in the cytosol. CA II inhibitors are emergent therapeutic targets for the development of antiglaucoma agents. Glaucoma is a disease affecting large number of people and characterized by an elevated intraocular pressure due to excessive secretion of aqueous humor. The enzyme CA II is responsible for the secretion of aqueous humor. Isoforms of CA II such as CA IV and CA I are present in many tissues and organs other than the eye. The commercially available CA inhibitors such as Brinzolamide tend to inhibit CA II and other isozymes such as CA IV and CA I. This leads to undesired side effects such as numbness and tingling of extremities, metallic taste, depression, fatigue, etc. In order to avoid these side effects an isozyme specific inhibitor is required. Here, we sought a novel DNA-based agent to inhibit specifically carbonic anhydrase II. Toward this goal, an 86 base pair, single-stranded DNA aptamer library containing a 40 base pair variable region was screened for CA II binding candidates, using ten rounds of positive and negative selection. Positive selection rounds has been used to evolve aptamers of extremely high binding affinity toward the target CA II whereas negative selection cycles were performed to determine the specificity of the aptamers toward CA II. Next generation sequencing (NGS) revealed sequences of the aptamer library. The most active anti-CAII aptamer had a random central sequence 5’-CGT AGC TAT GAT ATT GATT GTT TTT AAA TCC ACC CG TGCA -3’ and was designated as CA II inhibitory aptamer (CAII-A8). This aptamer was found to be isozyme specific inhibitor determined by the enzymatic assay of carbonic anhydrase for Wilbur Anderson Units (EC CAII-A8 is the first CA II isozyme specific inhibitory aptamer to be described as an antiglaucoma agent.

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Recommended Citation

Karim, Afroz, "Development of a DNA Aptamer that Specifically Inhibits Human Carbonic Anhydrase II" (2018). ETD Collection for University of Texas, El Paso. AAI10928636.