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dc.contributor.authorZhou, Wenhu
dc.contributor.authorZhang, Yupei
dc.contributor.authorHuang, Po-Jung Jimmy
dc.contributor.authorDing, Jinsong
dc.contributor.authorLiu, Juewen 16:12:01 (GMT) 16:12:01 (GMT)
dc.description.abstractMost 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. Ce3+), 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 Tb3+ 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, Tb3+ luminescence and DMS footprinting are 24.6, 16.3 and 47 mM, respectively. Mutation studies indicate that the role of Ce3+ might be replaced by G(23) in NaA43. Ce3+ functions by stabilizing the transition state phosphorane, thus promoting cleavage. G(23) competes favorably with low concentration Ce3+ (below 1 mu M). The G(23)-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.en
dc.description.sponsorshipNatural Sciences and Engineering Research Council of Canada (NSERC); Foundation for Shenghua Scholar of Central South University; National Natural Science Foundation of China [ 21301195]; Fellowship from the China Scholarship Council (CSC) [ 201406370116 to W.Z.]. Funding for open access charge: NSERC.en
dc.publisherOxford University Pressen
dc.rightsAttribution-NonCommercial 4.0 International*
dc.subjectLanthanide-Dependent Dnazymeen
dc.subjectIn-Vitro Selectionen
dc.subjectCleaving DNA Enzymeen
dc.subjectHammerhead Ribozymeen
dc.subjectCleavage Reactionen
dc.titleA DNAzyme requiring two different metal ions at two distinct sitesen
dcterms.bibliographicCitationZhou, W., Zhang, Y., Huang, P.-J. J., Ding, J., & Liu, J. (2016). A DNAzyme requiring two different metal ions at two distinct sites. Nucleic Acids Research, 44(1), 354–363.
uws.contributor.affiliation1Faculty of Scienceen
uws.contributor.affiliation3Waterloo Institute for Nanotechnology (WIN)en

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