@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.}
}
@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 = {<Go to ISI>://000226309400036},
	author = {Ting, R. and Thomas, J. M. and Lermer, L. and Perrin,David M.}
}
@article {430,
	title = {Toward an RNaseA mimic: a DNAzyme with imidazoles and cationic amines},
	journal = {Journal of the American Chemical Society},
	volume = {124},
	number = {34},
	year = {2002},
	note = {ISI Document Delivery No.: 586GJTimes Cited: 41Cited Reference Count: 52},
	month = {Aug},
	pages = {9960-9961},
	type = {Article},
	keywords = {CATALYTIC REPERTOIRE, CATIONS, CLEAVING DNA ENZYME, FUNCTIONALITIES, HAMMERHEAD, IN-VITRO SELECTION, MONOVALENT, NUCLEIC-ACIDS, RIBOZYME, SEQUENCE-SPECIFIC CLEAVAGE, SITE-SPECIFIC CLEAVAGE},
	isbn = {0002-7863},
	url = {<Go to ISI>://000177576000001},
	author = {Lermer, L. and Roupioz, Y. and Ting, R. and Perrin,David M.}
}
