@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 = {<Go to ISI>://000265268100049},
	author = {Thomas, J. M. and Yoon, J. K. and Perrin,David M.}
}
@article {2635,
	title = {Probing General Acid Catalysis in the Hammerhead Ribozyme},
	journal = {Journal of the American Chemical Society},
	volume = {131},
	number = {3},
	year = {2009},
	note = {ISI Document Delivery No.: 427PQTimes Cited: 5Cited Reference Count: 58Thomas, Jason M. Perrin, David M.},
	month = {Jan},
	pages = {1135-1143},
	type = {Article},
	abstract = {Recent crystallographic and computational studies have provided fresh insights into the catalytic mechanism of the RNA-cleaving hammerhead ribozyme. Based on these findings, specific ribozyme functional groups have been hypothesized to act directly as the general acid and base catalysts, although the catalytic role of divalent metal cations (M2+) remains uncertain: We now report a functional characterization of the general acid catalysis mechanism and the role of an M2+ cofactor therein, for the S. mansoni hammerhead (an "extended" hammerhead ribozyme). We have compared hammerhead cleavage of substrates with natural (ribo-phosphodiester) versus bridging-5{\textquoteright}-phosphorothioate scissile linkages, in the contexts of active site mutations and M2+ substitution. Cleavage of the natural substrate is inhibited by modification of the G8 2{\textquoteright}-OH ribozyme residue and depends strongly upon the presence and identity of an M2+ cofactor; in contrast, cleavage of the bridging-phosphorothioate substrate is conspicuously insensitive to any of these factors. These results imply that (1) both an M2+ cofactor and the G8 2{\textquoteright}-OH play crucial roles in hammerhead general acid catalysis and (2) the M2+ cofactor does not contribute to general acid catalysis via Lewis acid stabilization of the leaving group. General acid pK(a) perturbation was also demonstrated for both M2+ substitution and G8 2{\textquoteright}-OH modification, which suggests transition state M2+ coordination of the G8 2{\textquoteright}-OH, to lower its pK(a) and improve its ability to transfer a proton to the leaving group. We also report a simple method for synthesizing radiolabeled bridging-5{\textquoteright}-phosphorothioate substrates.},
	keywords = {2{\textquoteright}-HYDROXYL GROUP, ACTIVE-SITE, BASE CATALYSIS, CATIONS, MEDIATED CLEAVAGE, METAL-ION, MONOVALENT, P-31 NMR, PHOSPHOLIPASE-C, RIBONUCLEASE-A, SUBSTRATE-ANALOG},
	isbn = {0002-7863},
	url = {<Go to ISI>://000264791600054},
	author = {Thomas, J. M. and Perrin,David M.}
}
