@article {2438, title = {A DNAzyme with Three Protein-Like Functional Groups: Enhancing Catalytic Efficiency of M2+-Independent RNA Cleavage}, journal = {Chembiochem}, volume = {10}, number = {12}, year = {2009}, note = {ISI Document Delivery No.: 487MWTimes Cited: 5Cited Reference Count: 76Hollenstein, Marcel Hipolito, Christopher J. Lam, Curtis H. Perrin, David M.}, month = {Aug}, pages = {1988-1992}, type = {Article}, keywords = {BEACON SENSOR, biology, CLEAVING DNA ENZYME, CYTOSOLIC FREE MAGNESIUM, DEOXYRIBOZYMES, DNAzymes, enzyme catalysis, EVOLUTION, HAIRPIN RIBOZYME, HIGH-DENSITY, IN-VITRO SELECTION, ION, nucleic acids, NUCLEIC-ACIDS, RNAse A mimics, synthetic}, isbn = {1439-4227}, url = {://000269279700012}, author = {Hollenstein, M. and Hipolito, C. J. and Lam, C. H. and Perrin,David M.} } @article {2636, title = {Investigation of the Catalytic Mechanism of a Synthetic DNAzyme with Protein-like Functionality: An RNaseA Mimic?}, journal = {Journal of the American Chemical Society}, volume = {131}, number = {15}, year = {2009}, note = {ISI Document Delivery No.: 434IJTimes Cited: 3Cited Reference Count: 108Thomas, Jason M. Yoon, Jung-Ki Perrin, David M.}, month = {Apr}, pages = {5648-5658}, type = {Article}, abstract = {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.}, keywords = {ACID-BASE CATALYSIS, CLEAVING DNA ENZYME, COMPLEX, CYTOSOLIC FREE MAGNESIUM, DELTA VIRUS RIBOZYME, DINUCLEAR ZN(II), DIVALENT METAL-IONS, HAIRPIN RIBOZYME CATALYSIS, HDV GENOMIC, IN-VITRO SELECTION, RIBONUCLEASE-A, RIBOZYME}, isbn = {0002-7863}, url = {://000265268100049}, author = {Thomas, J. M. and Yoon, J. K. and Perrin,David M.} } @article {1017, title = {Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation}, journal = {Nucleic Acids Research}, volume = {32}, number = {22}, year = {2004}, note = {ISI Document Delivery No.: 887METimes Cited: 24Cited Reference Count: 71}, pages = {6660-6672}, type = {Article}, abstract = {This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9(25)-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9(25)-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5{\textquoteright}-product terminating in a 2{\textquoteright},3{\textquoteright}-phosphodiester and a 3{\textquoteright}-product terminating in a 5{\textquoteright}-OH. Under single turnover conditions at 24degreesC, 9(25)-11t displays a maximum first-order rate constant, k(cat), of 0.037 min(-1) and a catalytic efficiency, k(cat)/K-m, of 5.3 x 10(5) M-1 min(-1). The measured value of k(cat) under catalyst excess conditions agrees with the value of k(cat) observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9(25)-11t was gauged in terms of k(cat) values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k(cat) values by a factor of 4 to 250, indicating that 9(25)-11t displays significant sequence specificity despite the lack of an apparent Watson-Crick base-pairing scheme for recognition.}, keywords = {CATIONS, CLEAVING DNA ENZYME, CYTOSOLIC FREE MAGNESIUM, HAIRPIN RIBOZYME CLEAVAGE, HAMMERHEAD, IMIDAZOLYL FUNCTIONALITIES, IN-VITRO SELECTION, MONOVALENT, NUCLEIC-ACIDS, NUCLEOSIDE TRIPHOSPHATES, SEQUENCE-SPECIFIC CLEAVAGE}, isbn = {0305-1048}, url = {://000226309400036}, author = {Ting, R. and Thomas, J. M. and Lermer, L. and Perrin,David M.} }