title: Investigation of the catalytic mechanism of a synthetic DNAzyme with protein-like functionality: an RNaseA mimic?
abstract: The protein enzyme ribonuclease A (RNaseA) cleaves RNA with catalytic perfection, although with little sequence specificity, by a divalent metal ion (M(2+))-independent mechanism in which a pair of imidazoles provides general acid and base catalysis, while a cationic amine provides electrostatic stabilization of the transition state. Synthetic imitation of this remarkable organo-catalyst ("RNaseA mimicry") has been a longstanding goal in biomimetic chemistry. The 9(25)-11 DNAzyme contains synthetically modified nucleotides presenting both imidazole and cationic amine side chains, and catalyzes RNA cleavage with turnover in the absence of M(2+) similarly to RNaseA. Nevertheless, the catalytic roles, if any, of the "protein-like" functional groups have not been defined, and hence the question remains whether 9(25)-11 engages any of these functionalities to mimic aspects of the mechanism of RNaseA. To address this question, we report a mechanistic investigation of 9(25)-11 catalysis wherein we have employed a variety of experiments, such as DNAzyme functional group deletion, mechanism-based affinity labeling, and bridging and nonbridging phosphorothioate substitution of the scissile phosphate. Several striking parallels exist between the results presented here for 9(25)-11 and the results of analogous experiments applied previously to RNaseA. Specifically, our results implicate two particular imidazoles in general acid and base catalysis and suggest that a specific cationic amine stabilizes the transition state via diastereoselective interaction with the scissile phosphate. Overall, 9(25)-11 appears to meet the minimal criteria of an RNaseA mimic; this demonstrates how added synthetic functionality can expand the mechanistic repertoire available to a synthetic DNA-based catalyst.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, Canada V6T 1Z1.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Thomas
authors.forename: Jung-Ki
authors.initials: JK
authors.lastname: Yoon
authors.forename: David M
authors.initials: DM
authors.lastname: Perrin
mesh_terms: D000588:Amines; D002384:Catalysis; D021881:DNA, Catalytic / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D007093:Imidazoles; D018716:Molecular Mimicry; D010710:Phosphates; D012259:Ribonuclease, Pancreatic
chemical_list: D000588:Amines; D021881:DNA, Catalytic; C495395:DNAzyme, 9(25)-11t; D007093:Imidazoles; D010710:Phosphates; D012259:Ribonuclease, Pancreatic