title: In vitro selection and characterization of a novel Zn(II)-dependent phosphorothiolate thiolesterase ribozyme.
abstract: 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.
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. tzupinw@cc.kmu.edu.tw
authors.forename: Tzu-Pin
authors.initials: TP
authors.lastname: Wang
authors.forename: Yu-Chih
authors.initials: YC
authors.lastname: Su
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.forename: Yi-Ming
authors.initials: YM
authors.lastname: Liou
authors.forename: Kun-Liang
authors.initials: KL
authors.lastname: Lin
authors.forename: Eng-Chi
authors.initials: EC
authors.lastname: Wang
authors.forename: Long-Chih
authors.initials: LC
authors.lastname: Hwang
authors.forename: Yun-Ming
authors.initials: YM
authors.lastname: Wang
authors.forename: Yen-Hsu
authors.initials: YH
authors.lastname: Chen
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry / Q000235:genetics; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D006157:Guanosine Monophosphate / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics; D016337:RNA, Catalytic / Q000138:chemical synthesis* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013869:Thiolester Hydrolases / Q000138:chemical synthesis* / Q000235:genetics; D013873:Thionucleotides / Q000737:chemistry / Q000235:genetics; D015534:Trans-Activators / Q000138:chemical synthesis / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
chemical_list: D052157:Aptamers, Nucleotide; D016337:RNA, Catalytic; D013873:Thionucleotides; D015534:Trans-Activators; D001710:Biotin; C066524:guanosine 5'-monophosphorothioate; D006157:Guanosine Monophosphate; D009247:NADH, NADPH Oxidoreductases; C014703:disulfide reductase (NADH); D013869:Thiolester Hydrolases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing); D015032:Zinc