title: A DNAzyme requiring two different metal ions at two distinct sites.
abstract: Most previously reported RNA-cleaving DNAzymes require only a single divalent metal ion for catalysis. We recently reported a general trivalent lanthanide-dependent DNAzyme named Ce13d. This work shows that Ce13d requires both Na(+) and a trivalent lanthanide (e.g. Ce(3+)), simultaneously. This discovery is facilitated by the sequence similarity between Ce13d and a recently reported Na(+)-specific DNAzyme, NaA43. The Ce13d cleavage rate linearly depends on the concentration of both metal ions. Sensitized Tb(3+) luminescence and DMS footprinting experiments indicate that the guanines in the enzyme loop are important for Na(+)-binding. The Na(+) dissociation constants of Ce13d measured from the cleavage activity assay, Tb(3+) luminescence and DMS footprinting are 24.6, 16.3 and 47 mM, respectively. Mutation studies indicate that the role of Ce(3+) might be replaced by G23 in NaA43. Ce(3+) functions by stabilizing the transition state phosphorane, thus promoting cleavage. G23 competes favorably with low concentration Ce(3+) (below 1 μM). The G23-to-hypoxanthine mutation suggests the N1 position of the guanine as a hydrogen bond donor. Together, Ce13d has two distinct metal binding sites, each fulfilling a different role. DNAzymes can be quite sophisticated in utilizing metal ions for catalysis and molecular recognition, similar to protein metalloenzymes.
authors.affiliation: School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410013, China Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Wenhu
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Yupei
authors.initials: Y
authors.lastname: Zhang
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: School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410013, China.
authors.forename: Jinsong
authors.initials: J
authors.lastname: Ding
authors.affiliation: School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410013, China 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: D001483:Base Sequence; D002384:Catalysis; D002563:Cerium / Q000737:chemistry; D021881:DNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D019143:Evolution, Molecular; D007477:Ions / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D012964:Sodium / Q000737:chemistry
chemical_list: D021881:DNA, Catalytic; D007477:Ions; D008670:Metals; D002563:Cerium; D012964:Sodium