An alcohol dehydrogenase ribozyme: Evolution, characterization and reengineering
Pattnaik, Swetansu B.
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I report a ribonucleic acid (RNA) molecule that exhibits activity analogous to that of alcohol dehydrogenase (ADH). Directed in vitro evolution was used to enrich nicotinamide adenine dinucleotide (NAD + )--dependent redox-active RNAs from a combinatorial pool. The most active ribozyme in the population forms a compact pseudoknotted structure and oxidizes an alcohol seven orders of magnitude faster than the estimated spontaneous rate. Moreover, this ADH RNA was coupled with a redox relay between NADH and flavin adenine dinucleotide to give a NAD + -regeneration system. I have also demonstrated that the redox active ribozyme is able to reduce an aldehyde assisted by NADH/Zn 2+ . Although the reversibility of phosphodiester and acyl transfer reactions catalyzed by ribozymes was known, that of other chemical reactions has not been well established. This study has demonstrated the reversibility of hydride transfer chemistry, involving C-H bond formation/cleavage, catalyzed by the ADH ribozyme. Though its exact mechanism remains to be determined, the fact that the ribozyme shares many features with the protein ADHs e.g., reversibility and NADH/Zn 2+ -dependence, makes this RNA-based redox system intriguing. This demonstration of the redox ability of RNA adds support to an RNA-based metabolic system in ancient life. In addition, I characterized the ribozyme structure and identified the structural elements contributing towards enhancing the catalytic ability of the ribozyme. These studies have been very insightful in understanding some intricate relationships between structure and function in a less speculative manner.