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RNA aptamer inhibitors of a restriction endonuclease
Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding pr...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551934/ https://www.ncbi.nlm.nih.gov/pubmed/26184872 http://dx.doi.org/10.1093/nar/gkv702 |
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author | Mondragón, Estefanía Maher, L. James |
author_facet | Mondragón, Estefanía Maher, L. James |
author_sort | Mondragón, Estefanía |
collection | PubMed |
description | Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (K(d) ∼12-30 nM) selective competitive inhibitors (IC(50) ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors. |
format | Online Article Text |
id | pubmed-4551934 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-45519342015-08-28 RNA aptamer inhibitors of a restriction endonuclease Mondragón, Estefanía Maher, L. James Nucleic Acids Res RNA Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (K(d) ∼12-30 nM) selective competitive inhibitors (IC(50) ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors. Oxford University Press 2015-09-03 2015-07-15 /pmc/articles/PMC4551934/ /pubmed/26184872 http://dx.doi.org/10.1093/nar/gkv702 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | RNA Mondragón, Estefanía Maher, L. James RNA aptamer inhibitors of a restriction endonuclease |
title | RNA aptamer inhibitors of a restriction endonuclease |
title_full | RNA aptamer inhibitors of a restriction endonuclease |
title_fullStr | RNA aptamer inhibitors of a restriction endonuclease |
title_full_unstemmed | RNA aptamer inhibitors of a restriction endonuclease |
title_short | RNA aptamer inhibitors of a restriction endonuclease |
title_sort | rna aptamer inhibitors of a restriction endonuclease |
topic | RNA |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551934/ https://www.ncbi.nlm.nih.gov/pubmed/26184872 http://dx.doi.org/10.1093/nar/gkv702 |
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