In Vitro Selection and Characterization of a Novel Zn(II)-Dependent Phosphorothiolate Thiolesterase Ribozyme

Here we present the in vitro selection of a novel ribozyme specific for Zn²⁺-dependent catalysis on hydrolysis of a phosphorothiolate thiolester bond. The ribozyme, called the TW17 ribozyme, was evolved and selected from an artificial RNA pool covalently linked to a biotin-containing substrate through the phosphorothiolate thiolester bond. The secondary structure for the evolved ribozyme consisted of three major helices and three loops. Biochemical and chemical studies of ribozyme-catalyzed reaction products provided evidence that the ribozyme specifically catalyzes hydrolysis of the phosphorothiolate thiolester linkage. A successful ribozyme construct with active catalysis in trans further supported the determined ribozyme structure and indicated the potential of the ribozyme for multiple-substrate turnover. The ribozyme also requires Zn²⁺ and Mg²⁺ for maximal catalysis. The TW17 ribozyme, in the presence of Zn²⁺ and Mg²⁺, conferred a rate enhancement of at least 5 orders of magnitude when compared to the estimated rate of the uncatalyzed reaction. The ribozyme completely lost catalytic activity in the absence of Zn²⁺, like Zn²⁺-dependent protein hydrolases. The discovery and characterization of the TW17 ribozyme suggest additional roles for Zn²⁺ in ribozyme catalysts.