Water as a specific acid/base catalyst in the mechanism Panel 1 Water as a specific acid/base catalyst in the mechanism of action of a non-coding catalytic RNA Joseph E. Wedekind, Associate Professor Department of Biochemistry & Biophysics, University of Rochester RNA enzymes catalyze important biological reactions, yet little is known of the principles they employ to achieve such activity. This catalytic conundrum is complicated by a lack of reactive chemical groups within the RNA polymer suited for rate acceleration under mild, physiological conditions. Our prior work led to the hypothesis that water molecules play important roles in the reaction of the hairpin ribozyme – a model RNA enzyme derived from subviral plant pathogens. Using X-ray crystallography, we determined structures of the hairpin ribozyme (Panel 1) in complex with analogs that mimic conformations along the reaction path (Panel 2). The results revealed two water molecules that appear to stabilize the transition-state relative to substrate- and product-bound states. Panel 2 We have also succeeded in the chemical syntheses of a 5’-deoxy-5’-fluoro analog of G+1 and an N1 deaza analog of A38 that are each being used to prepare more accurate mimics of the product complex (red & blue boxes, respectively). Future efforts will focus on syntheses of analogs that displace specific waters, which should inhibit catalysis if the molecule is essential for activity. Overall the results provide new insight into the importance of water in RNA mediated reactions and the principles that ribozymes employ to achieve biological functionality.