title: Searching for a DNAzyme Version of the Leadzyme.
abstract: The leadzyme refers to a small ribozyme that cleaves a RNA substrate in the presence of Pb(2+). In an optimized form, the enzyme strand contains only two unpaired nucleotides. Most RNA-cleaving DNAzymes are much longer. Two classical Pb(2+)-dependent DNAzymes, 8-17 and GR5, both contain around 15 nucleotides in the enzyme loop. This is also the size of most RNA-cleaving DNAzymes that use other metal ions for their activity. Such large enzyme loops make spectroscopic characterization difficult and so far no high-resolution structural information is available for active DNAzymes. The goal of this work is to search for DNAzymes with smaller enzyme loops. A simple replacement of the ribonucleotides in the leadzyme by deoxyribonucleotides failed to produce an active enzyme. A Pb(2+)-dependent in vitro selection combined with deep sequencing was then performed. After sequence alignment and DNA folding, a new DNAzyme named PbE22 was identified, which contains only 5 nucleotides in the enzyme catalytic loop. The biochemical characteristics of PbE22 were compared with those of the leadzyme and the two classical Pb(2+)-dependent DNAzymes. The rate of PbE22 rises with increase in Pb(2+) concentration, being 1.7 h(-1) in the presence of 100 μM Pb(2+) and reaching 3.5 h(-1) at 500 µM Pb(2+). The log of PbE22 rate rises linearly in a pH-dependent fashion (20 µM Pb(2+)) with a slope of 0.74. In addition, many other abundant sequences in the final library were studied. These sequences are quite varied in length and nucleotide composition, but some contain a few conserved nucleotides consistent with the GR5 structure. Interestingly, some sequences are active with Pb(2+) but none of them were active with even 50 mM Mg(2+), which is reminiscent of the difference between the GR5 and 8-17 DNAzymes.
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON, N2L 3G1, Canada.
authors.forename: Runjhun
authors.initials: R
authors.lastname: Saran
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON, N2L 3G1, Canada.
authors.forename: Qingyun
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON, N2L 3G1, Canada. liujw@uwaterloo.ca.
authors.forename: Juewen
authors.initials: J
authors.lastname: Liu
mesh_terms: D020134:Catalytic Domain; D021881:DNA, Catalytic / Q000378:metabolism*; D007700:Kinetics; D059372:Nucleotide Motifs; D016337:RNA, Catalytic / Q000378:metabolism*; D052156:SELEX Aptamer Technique
chemical_list: D021881:DNA, Catalytic; D016337:RNA, Catalytic; C113411:leadzyme
keywords: DNAzymes; In vitro selection; Lead; RNA cleavage; Ribozymes