Corroboration of Zn(ii)-Mg(ii)-tertiary structure interplays essential for the optimal catalysis of a...

The TW17 ribozyme, a catalytic RNA selected from a pool of artificial RNA, is specific for the Zn²⁺-dependent hydrolysis of a phosphorothiolate thiolester bond. Here, we describe the organic synthesis of both guanosine α-thio-monophosphate and the substrates required for selecting and characterizing the TW17 ribozyme, and for deciphering the catalytic mechanism of the ribozyme. By successively substituting the substrate originally conjugated to the RNA pool with structurally modified substrates, we demonstrated that the TW17 ribozyme specifically catalyzes phosphorothiolate thiolester hydrolysis. Metal titration studies of TW17 ribozyme catalysis in the presence of Zn²⁺ alone, Zn²⁺ and Mg²⁺, and Zn²⁺ and [Co(NH₃)₆]³⁺ supported our findings that Zn²⁺ is absolutely required for ribozyme catalysis, and indicated that optimal ribozyme catalysis involves the presence of outer-sphere and one inner-sphere Mg²⁺. A survey of the TW17 ribozyme activity at various pHs revealed that the activity of the ribozyme critically depends on the alkaline conditions. Moreover, a GNRA tetraloop-containing ribozyme constructed with active catalysis in trans provided catalysis and multiple substrate turnover efficiencies significantly higher than ribozymes lacking a GNRA tetraloop. This research supports the essential roles of Zn²⁺, Mg²⁺, and a GNRA tetraloop in modulating the TW17 ribozyme structure for optimal ribozyme catalysis, leading also to the formulation of a proposed reaction mechanism for TW17 ribozyme catalysis.