The protein enzyme ribonuclease A (RNaseA) cleaves RNA with catalytic perfection, although with little sequence specificity, by a divalent metal ion (M2+)-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 M2+ similarly to RNaseA. Nevertheless, the catalytic roles, if any, of the "proteinlike" functional groups have not been defined, and hence the question remains whether 925-11 engages any of these functionalities to mimic aspects of the mechanism of RNaseA. To address this question, we report a mechanistic investigation Of 925-11 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 925-11 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, 925-11 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.