title: Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme.
abstract: BACKGROUND
  RNA and DNA are polymers that lack the diversity of chemical functionalities that make proteins so suited to biological catalysis. All naturally occurring ribozymes (RNA catalysts) that catalyze the formation, transfer and hydrolysis of phosphodiesters require metal-ion cofactors for their catalytic activity. We wished to investigate whether, and to what extent, DNA molecules could catalyze the cleavage (by either hydrolysis or transesterification) of a ribonucleotide phosphodiester in the absence of divalent or higher-valent metal ions or, indeed, any other cofactors.
  
  
  RESULTS
  We performed in vitro selection and amplification experiments on a library of random-sequence DNA that incorporated a single ribonucleotide, a suitable site for cleavage. Following 12 cycles of selection and amplification, a 'first generation' of DNA enzymes (DNAzymes) cleaved their internal ribonucleotide phosphodiesters at rates approximately 10(7)-fold faster than the spontaneous rate of cleavage of the dinucleotide ApA in the absence of divalent cations. Re-selection from a partially randomized DNA pool yielded 'second generation' DNAzymes that self-cleaved at rates of approximately 0.01 min-1 (a 10(8)-fold rate enhancement over the cleavage rate of ApA). The properties of these selected catalysts were different in key respects from those of metal-utilizing ribozymes and DNAzymes. The catalyzed cleavage took place in the presence of different chelators and ribonuclease inhibitors. Trace-metal analysis of the reaction buffer (containing very high purity reagents) by inductively coupled plasma-optical emission spectrophotometry indicated that divalent or higher-valent metal ions do not mediate catalysis by the DNAzymes.
  
  
  CONCLUSIONS
  Our results indicate that, although ribozymes are sometimes regarded generically to be metalloenzymes, the nucleic acid components of ribozymes may play a substantial role in the overall catalysis. Given that metal cofactors increase the rate of catalysis by ribozymes only approximately 10(2)-10(3)-fold above that of the DNAzyme described in this paper, it is conceivable that substrate positioning, transition-state stabilization or general acid/base catalysis by the nucleic acid components of ribozymes and DNAzymes may contribute significantly to their overall catalytic performance.
authors.affiliation: Institute of Molecular Biology and Biochemistry, Simon Fraser University Burnaby, BC, Canada V5A 1S6.
authors.forename: C R
authors.initials: CR
authors.lastname: Geyer
authors.forename: D
authors.initials: D
authors.lastname: Sen
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002021:Buffers; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded / Q000378:metabolism*; D066298:In Vitro Techniques; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
chemical_list: D002021:Buffers; D003067:Coenzymes; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D008670:Metals; D004247:DNA