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A DNAzyme requiring two different metal ions at two distinct sites

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 disc...

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Autores principales: Zhou, Wenhu, Zhang, Yupei, Huang, Po-Jung Jimmy, Ding, Jinsong, Liu, Juewen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705669/
https://www.ncbi.nlm.nih.gov/pubmed/26657636
http://dx.doi.org/10.1093/nar/gkv1346
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author Zhou, Wenhu
Zhang, Yupei
Huang, Po-Jung Jimmy
Ding, Jinsong
Liu, Juewen
author_facet Zhou, Wenhu
Zhang, Yupei
Huang, Po-Jung Jimmy
Ding, Jinsong
Liu, Juewen
author_sort Zhou, Wenhu
collection PubMed
description 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 G(23) in NaA43. Ce(3+) functions by stabilizing the transition state phosphorane, thus promoting cleavage. G(23) competes favorably with low concentration Ce(3+) (below 1 μM). 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.
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spelling pubmed-47056692016-01-11 A DNAzyme requiring two different metal ions at two distinct sites Zhou, Wenhu Zhang, Yupei Huang, Po-Jung Jimmy Ding, Jinsong Liu, Juewen Nucleic Acids Res Nucleic Acid Enzymes 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 G(23) in NaA43. Ce(3+) functions by stabilizing the transition state phosphorane, thus promoting cleavage. G(23) competes favorably with low concentration Ce(3+) (below 1 μM). 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. Oxford University Press 2016-01-08 2015-12-10 /pmc/articles/PMC4705669/ /pubmed/26657636 http://dx.doi.org/10.1093/nar/gkv1346 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Nucleic Acid Enzymes
Zhou, Wenhu
Zhang, Yupei
Huang, Po-Jung Jimmy
Ding, Jinsong
Liu, Juewen
A DNAzyme requiring two different metal ions at two distinct sites
title A DNAzyme requiring two different metal ions at two distinct sites
title_full A DNAzyme requiring two different metal ions at two distinct sites
title_fullStr A DNAzyme requiring two different metal ions at two distinct sites
title_full_unstemmed A DNAzyme requiring two different metal ions at two distinct sites
title_short A DNAzyme requiring two different metal ions at two distinct sites
title_sort dnazyme requiring two different metal ions at two distinct sites
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705669/
https://www.ncbi.nlm.nih.gov/pubmed/26657636
http://dx.doi.org/10.1093/nar/gkv1346
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