Journal
NUCLEIC ACIDS RESEARCH
Volume 43, Issue 1, Pages 461-469Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gku1296
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Funding
- Canadian Institutes of Health Research (CIHR)
- Ontario Ministry of Research Innovation
- Natural Sciences and Engineering Research Council (NSERC) of Canada (Discovery Grant and Strategic Project Grant)
- CIHR
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In vitro selection of RNA-cleaving DNAzymes was performed using three heavy lanthanide ions (Ln(3+)): Ho3+, Er3+ and Tm3+. The resulting sequences were aligned together and about half of the library contained a new family of DNAzyme. These DNAzymes have a simple loop structure, and they are active only with the seven heavy Ln(3+). Among the tested non-lanthanide ions, only Y3+ induced cleavage and even Pb2+ failed to cleave, suggesting a very high specificity. A representative DNAzyme, Tm7, has a sigmoidal metal binding curve with a Hill coefficient of 3, indicating that three metal ions are involved in the catalytic step. Its pH-rate profile has a slope of 1, suggesting a single deprotonation step is involved in the rate-limiting step. Tm7 has a cleavage rate of 1.6 min(-1) at pH 7.8 with 10 mu M Er3+. Phosphorothioate substitution at the cleavage junction completely inhibits the activity, which cannot be rescued by Cd2+ alone, or by a mixture of Er3+ and Cd2+, suggesting that two interacting metal ions are involved in direct bonding to both non-bridging oxygen atoms. A new model involving three lanthanide ions is proposed based on this study. A biosensor is engineered using Tm7 to detect Dy3+ down to 14 nM.
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