NDLI: Searching for a DNAzyme Version of the Leadzyme

Content Provider	Springer Nature Link
Author	Saran, Runjhun Chen, Qingyun Liu, Juewen
Copyright Year	2015
Abstract	The leadzyme refers to a small ribozyme that cleaves a RNA substrate in the presence of Pb2+. In an optimized form, the enzyme strand contains only two unpaired nucleotides. Most RNA-cleaving DNAzymes are much longer. Two classical Pb2+-dependent DNAzymes, 8–17 and GR5, both contain around 15 nucleotides in the enzyme loop. This is also the size of most RNA-cleaving DNAzymes that use other metal ions for their activity. Such large enzyme loops make spectroscopic characterization difficult and so far no high-resolution structural information is available for active DNAzymes. The goal of this work is to search for DNAzymes with smaller enzyme loops. A simple replacement of the ribonucleotides in the leadzyme by deoxyribonucleotides failed to produce an active enzyme. A Pb2+-dependent in vitro selection combined with deep sequencing was then performed. After sequence alignment and DNA folding, a new DNAzyme named PbE22 was identified, which contains only 5 nucleotides in the enzyme catalytic loop. The biochemical characteristics of PbE22 were compared with those of the leadzyme and the two classical Pb2+-dependent DNAzymes. The rate of PbE22 rises with increase in Pb2+ concentration, being 1.7 h−1 in the presence of 100 μM Pb2+ and reaching 3.5 h−1 at 500 µM Pb2+. The log of PbE22 rate rises linearly in a pH-dependent fashion (20 µM Pb2+) with a slope of 0.74. In addition, many other abundant sequences in the final library were studied. These sequences are quite varied in length and nucleotide composition, but some contain a few conserved nucleotides consistent with the GR5 structure. Interestingly, some sequences are active with Pb2+ but none of them were active with even 50 mM Mg2+, which is reminiscent of the difference between the GR5 and 8–17 DNAzymes.
Starting Page	235
Ending Page	244
Page Count	10
File Format	PDF
ISSN	00222844
Journal	Journal of Molecular Evolution
Volume Number	81
Issue Number	5-6
e-ISSN	14321432
Language	English
Publisher	Springer US
Publisher Date	2015-10-12
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	DNAzymes RNA cleavage Lead Ribozymes In vitro selection Evolutionary Biology Microbiology Plant Sciences Plant Genetics & Genomics Animal Genetics and Genomics Cell Biology
Content Type	Text
Resource Type	Article
Subject	Genetics Molecular Biology Ecology, Evolution, Behavior and Systematics

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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