Project Summary Our long-term goal is to characterize the structure and activity of proteins in the eye that are associated with glaucoma pathogenesis, and thus present novel treatment strategies and therapeutics for glaucoma. The objective of the current project is to develop isozyme-selective inhibitors against extracellular carbonic anhydrase (CA) isozymes that are expressed in the eye, namely CA9, CA12, and CA14. These inhibitors will enable dissection of the contribution of different CA isozymes to glaucoma and thereby identify the best target protein for anti-glaucoma drug development. We propose to develop isozyme- selective CA inhibitors using aptamer technology. Aptamers are short oligonucleotides that bind to a wide range of biological molecules with high affinity and specificity. Aptamers are typically generated using a combinatorial technique called systematic evolution of ligands by exponential enrichment (SELEX). However, traditional SELEX selects aptamer sequences which are binders and not necessarily inhibitors. To accommodate our need, we have developed a novel SELEX scheme, called “expanded SELEX (eSELEX)”, which can generate isozyme-selective inhibitors against virtually any isozyme family.