Reengineering aninvitroevolved ribozyme.

Ribox02 is an in-vitro evolved RNA molecule that exhibits the activity analogous to that of alcohol dehydrogenase, catalyzing redox reactions in a cofactor dependent manner. It forms a compact pseudo-knotted structure and oxidizes an alcohol seven orders of magnitude faster than the estimated spontaneous rate. To gain insights into the structure-function relationship of Ribox02, we firstly performed mutational studies to identify the critical nucleotide bases for catalysis and then engineered the ribozyme structure to improve its overall fold. The re-engineered Ribox02, named Robust, was able to catalyze the reaction approximately 25% more efficiently, leading us to generate a transacting system.