dc.contributor.author | Saran, Runjhun | |
dc.contributor.author | Chen, Qingyun | |
dc.contributor.author | Liu, Juewen | |
dc.date.accessioned | 2017-02-23 21:04:34 (GMT) | |
dc.date.available | 2017-02-23 21:04:34 (GMT) | |
dc.date.issued | 2015-10-12 | |
dc.identifier.uri | http://dx.doi.org/10.1007/s00239-015-9702-z | |
dc.identifier.uri | http://hdl.handle.net/10012/11346 | |
dc.description | The final publication is available at Springer via http://dx.doi.org/10.1007/s00239-015-9702-z | en |
dc.description.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. | en |
dc.description.sponsorship | University of Waterloo ||
Ontario Ministry of Research & Innovation ||
Natural Sciences and Engineering Research Council || | en |
dc.language.iso | en | en |
dc.publisher | Springer | en |
dc.subject | DNAzymes | en |
dc.subject | RNA cleavage | en |
dc.subject | Lead | en |
dc.subject | Ribozymes | en |
dc.subject | In vitro selection | en |
dc.title | Searching for a DNAzyme version of the leadzyme | en |
dc.type | Article | en |
dcterms.bibliographicCitation | Saran, R., Chen, Q., & Liu, J. (2015). Searching for a DNAzyme Version of the Leadzyme. Journal of Molecular Evolution, 81(5–6), 235–244. https://doi.org/10.1007/s00239-015-9702-z | en |
uws.contributor.affiliation1 | Faculty of Science | en |
uws.contributor.affiliation2 | Chemistry | en |
uws.contributor.affiliation2 | Waterloo Institute for Nanotechnology (WIN) | en |
uws.typeOfResource | Text | en |
uws.peerReviewStatus | Reviewed | en |
uws.scholarLevel | Faculty | en |