@article {2285, title = {Probing General Base Catalysis in the Hammerhead Ribozyme}, journal = {Journal of the American Chemical Society}, volume = {130}, number = {46}, year = {2008}, note = {ISI Document Delivery No.: 406QZTimes Cited: 8Cited Reference Count: 53Thomas, Jason M. Perrin, David M.}, month = {Nov}, pages = {15467-15475}, type = {Article}, abstract = {Recent structural and computational studies have shed new light on the catalytic mechanism and active site structure of the RNA cleaving hammerhead ribozyme. Consequently, specific ribozyme functional groups have been hypothesized to be directly involved in general/acid base catalysis. In order to test this hypothesis, we have developed an affinity label to identify the functional general base in the S. mansoni hammerhead ribozyme. The ribozyme was reacted with a substrate analogue bearing a 2{\textquoteright}-bromoacetamide group in place of the nucleophilic 2{\textquoteright}-hydroxyl group which would normally be deprotonated by a general base. The electrophilic 2{\textquoteright}-bromoacetamide group is poised to alkylate the general base, which is subsequently identified by footprinting analysis. Herein, we demonstrate alkylation of N1 of G12 in the hammerhead ribozyme in a pH and [Mg2+] dependent manner that is consistent with the native cleavage reaction. These results provide substantial evidence that deprotonated N1 of G12 functions directly as a general base in the hammerhead ribozyme; moreover, our experiments provide evidence that the pK(a) of G12 is perturbed downward in the context of the active site structure. We also observed other pH-independent alkylations, which do not appear to reflect the catalytic mechanism, but offer further insight into ribozyme conformation and structure.}, keywords = {ACTIVE-SITE, CLEAVAGE, CRYSTAL-STRUCTURE, DELTA VIRUS RIBOZYME, HAIRPIN RIBOZYME, MECHANISM, METAL-ION, MONOVALENT CATIONS, NUCLEOBASE CATALYSIS, RNA}, isbn = {0002-7863}, url = {://000263311300051}, author = {Thomas, J. M. and Perrin,David M.} } @article {429, title = {Incorporation of 8-histaminyldeoxyadenosine [8-(2-(4-imidazolyl)ethylamino)-2 {\textquoteright}-deoxyriboadenosine] into oligodeoxyribonucleotides by solid phase phosphoramidite coupling}, journal = {Nucleosides Nucleotides \& Nucleic Acids}, volume = {21}, number = {10}, year = {2002}, note = {ISI Document Delivery No.: 622CATimes Cited: 5Cited Reference Count: 37}, pages = {651-664}, type = {Article}, abstract = {The 3{\textquoteright} phosphoramidite of 8-histaminyl deoxyadenosine has been prepared and successfully incorporated into a short oligodeoxyribonucleotide. The synthetic methodology leading to this preparation is given and the implications for developing new DNAzymes as well as probing unusual nucleic acid structures are discussed.}, keywords = {BEARING IMIDAZOLE, CATALYTIC REPERTOIRE, CLEAVAGE, DERIVATIVES, DNA, IMIDAZOLYL FUNCTIONALITIES, NUCLEIC-ACIDS, OLIGONUCLEOTIDES, RIBONUCLEASES, RNA}, isbn = {1525-7770}, url = {://000179627900003}, author = {Lermer, L. and Hobbs, J. and Perrin,David M.} }