title: In Vitro Selection of a DNAzyme Cooperatively Binding Two Lanthanide Ions for RNA Cleavage.
abstract: Trivalent lanthanide ions (Ln(3+)) were recently employed to select RNA-cleaving DNAzymes, and three new DNAzymes have been reported so far. In this work, dysprosium (Dy(3+)) was used with a library containing 50 random nucleotides. After six rounds of in vitro selection, a new DNAzyme named Dy10a was obtained and characterized. Dy10a has a bulged hairpin structure cleaving a RNA/DNA chimeric substrate. Dy10a is highly active in the presence of the five Ln(3+) ions in the middle of the lanthanide series (Sm(3+), Eu(3+), Gd(3+), Tb(3+), and Dy(3+)), while its activity descends on the two sides. The cleavage rate reaches 0.6 min(-1) at pH 6 with just 200 nM Sm(3+), which is the fastest among all known Ln(3+)-dependent enzymes. Dy10a binds two Ln(3+) ions cooperatively. When a phosphorothioate (PS) modification is introduced at the cleavage junction, the activity decreases by >2500-fold for both the Rp and Sp diastereomers, and thiophilic Cd(2+) cannot rescue the activity. The pH-rate profile has a slope of 0.37 between pH 4.2 and 5.2, and the slope was even lower at higher pH. On the basis of these data, a model of metal binding is proposed. Finally, a catalytic beacon sensor that can detect Ho(3+) down to 1.7 nM is constructed.
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo , 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
authors.forename: Po-Jung Jimmy
authors.initials: PJ
authors.lastname: Huang
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo , 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
authors.forename: Mahsa
authors.initials: M
authors.lastname: Vazin
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo , 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
authors.forename: Juewen
authors.initials: J
authors.lastname: Liu
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D021881:DNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D028581:Lanthanoid Series Elements / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D059367:RNA Cleavage / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D021881:DNA, Catalytic; D028581:Lanthanoid Series Elements