A new and efficient DNA enzyme for the sequence-specific cleavage of RNA.
A new DNA enzyme, the "Bipartite DNAzyme", suitable for the sequence-specific cleavage of RNA, was obtained from a random DNA library by in vitro selection. Only a single family of catalytic molecules emerged from the selection, and a 22 nucleotide consensus sequence common to all clones defined a putative catalytic core. The most abundant clone self-cleaved at a single internal ribonucleotide phosphodiester with a relatively fast k(obs) value of 1.7 min(-1), in 10 mM MgCl(2) at 23 degrees C. This DNAzyme ("Bipartite I") required divalent cations, with magnesium and manganese most optimally supporting cleavage. A reselection from a mutagenized DNAzyme pool for the ability to cleave at extended RNA substrates yielded an unchanged catalytic core sequence. From this re-selection a DNAzyme ("Bipartite II") capable of sequence-specifically cleaving extended stretches of RNA was derived. A rate versus pH analysis of the Bipartite II DNAzyme revealed a two-phase profile, similar to that reported for the hepatitis delta virus (HDV) ribozyme, suggesting that the Bipartite II DNAzyme and the HDV ribozyme may share similar catalytic strategies. Multiple-turnover kinetics, measured in 30 mM MgCl(2), at 37 degrees C, with an HIV-1-derived RNA substrate, yielded a k(cat) value of approximately 1.4 min(-1) and a K(M) value of approximately 230 nM, which were of the same order as k(cat) and K(M )values measured for other ribozymes and DNAzymes in general use for RNA cleavage. The Bipartite DNAzyme therefore represents a new and potentially useful reagent, both for the processing of RNA transcripts in vitro and for mRNA ablation procedures in vivo.
Feldman
A R
AR
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada.
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D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D021881:DNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D015686:Gene Products, env / Q000235:genetics; D006678:HIV / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D021881:DNA, Catalytic; D015686:Gene Products, env; D009843:Oligoribonucleotides; D015636:Magnesium Chloride; D012313:RNA