title: Rational evolution of Cd2+-specific DNAzymes with phosphorothioate modified cleavage junction and Cd2+ sensing.
abstract: In vitro selection of RNA-cleaving DNAzymes is a powerful method for isolating metal-specific DNA. A few successful examples are known, but it is still difficult to target some thiophilic metals such as Cd(2+) due to limited functional groups in DNA. While using modified bases expands the chemical functionality of DNA, a single phosphorothioate modification might boost its affinity for thiophilic metals without complicating the selection process or using bases that are not commercially available. In this work, the first such in vitro selection for Cd(2+) is reported. After using a blocking DNA and negative selections to rationally direct the library outcome, a highly specific DNAzyme with only 12 nucleotides in the catalytic loop is isolated. This DNAzyme has a cleavage rate of 0.12 min(-1) with 10 μM Cd(2+) at pH 6.0. The Rp form of the substrate is cleaved ∼100-fold faster than the Sp form. The DNAzyme is most active with Cd(2+) and its selectivity against Zn(2+) is over 100 000-fold. Its application in detecting Cd(2+) is also demonstrated. The idea of introducing single modifications in the fixed region expands the scope of DNA/metal interactions with minimal perturbation of DNA structure and property.
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Po-Jung Jimmy
authors.initials: PJ
authors.lastname: Huang
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada liujw@uwaterloo.ca.
authors.forename: Juewen
authors.initials: J
authors.lastname: Liu
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D002104:Cadmium / Q000032:analysis* / Q000378:metabolism; D021881:DNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D019020:Directed Molecular Evolution; D015723:Gene Library; D009690:Nucleic Acid Conformation; D012275:Oryza / Q000737:chemistry; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry*; D059367:RNA Cleavage; D013237:Stereoisomerism
chemical_list: D021881:DNA, Catalytic; D054735:Phosphorothioate Oligonucleotides; D002104:Cadmium