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High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication
HIV-1 aspartyl protease (PR) plays a key role in virion morphogenesis, underscoring the effectiveness of protease inhibitors (PI). Despite their utility, side effects and drug-resistance remains a problem. We report the development of RNA aptamers as inhibitors of HIV-1 PR for potential use in anti-...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345311/ https://www.ncbi.nlm.nih.gov/pubmed/25689224 http://dx.doi.org/10.1038/mtna.2015.1 |
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author | Duclair, Sonald Gautam, Archana Ellington, Andrew Prasad, Vinayaka R |
author_facet | Duclair, Sonald Gautam, Archana Ellington, Andrew Prasad, Vinayaka R |
author_sort | Duclair, Sonald |
collection | PubMed |
description | HIV-1 aspartyl protease (PR) plays a key role in virion morphogenesis, underscoring the effectiveness of protease inhibitors (PI). Despite their utility, side effects and drug-resistance remains a problem. We report the development of RNA aptamers as inhibitors of HIV-1 PR for potential use in anti-HIV gene therapy. Employing Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated four unique families of anti-HIV-1 PR RNA aptamers displaying moderate binding affinities (K(d) = 92–140 nmol/l) and anti-PR inhibitory activity (K(i)s = 138–647 nmol/l). Second-generation RNA aptamers selected from partially randomized pools based on two of the aptamer sequences displayed striking enhancements in binding (K(d)s = 2–22 nmol/l) and inhibition (K(i)s = 31–49 nmol/l). The aptamers were specific in that they did not bind either the related HIV-2 protease, or the cellular aspartyl protease, Cathepsin D. Site-directed mutagenesis of a second-generation aptamer to probe the predicted secondary structure indicated that the stem-loops SL2 and SL3 and the stem P1 were essential for binding and that only the 3'-most 17 nucleotides were dispensable. Anti-PR aptamers inhibited HIV replication in vitro and the degree of inhibition was higher for second-generation aptamers with greater affinity and the inhibition was abrogated for a nonbinding aptamer variant. |
format | Online Article Text |
id | pubmed-4345311 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-43453112015-03-10 High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication Duclair, Sonald Gautam, Archana Ellington, Andrew Prasad, Vinayaka R Mol Ther Nucleic Acids Original Article HIV-1 aspartyl protease (PR) plays a key role in virion morphogenesis, underscoring the effectiveness of protease inhibitors (PI). Despite their utility, side effects and drug-resistance remains a problem. We report the development of RNA aptamers as inhibitors of HIV-1 PR for potential use in anti-HIV gene therapy. Employing Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated four unique families of anti-HIV-1 PR RNA aptamers displaying moderate binding affinities (K(d) = 92–140 nmol/l) and anti-PR inhibitory activity (K(i)s = 138–647 nmol/l). Second-generation RNA aptamers selected from partially randomized pools based on two of the aptamer sequences displayed striking enhancements in binding (K(d)s = 2–22 nmol/l) and inhibition (K(i)s = 31–49 nmol/l). The aptamers were specific in that they did not bind either the related HIV-2 protease, or the cellular aspartyl protease, Cathepsin D. Site-directed mutagenesis of a second-generation aptamer to probe the predicted secondary structure indicated that the stem-loops SL2 and SL3 and the stem P1 were essential for binding and that only the 3'-most 17 nucleotides were dispensable. Anti-PR aptamers inhibited HIV replication in vitro and the degree of inhibition was higher for second-generation aptamers with greater affinity and the inhibition was abrogated for a nonbinding aptamer variant. Nature Publishing Group 2015-02 2015-02-17 /pmc/articles/PMC4345311/ /pubmed/25689224 http://dx.doi.org/10.1038/mtna.2015.1 Text en Copyright © 2015 American Society of Gene & Cell Therapy http://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ |
spellingShingle | Original Article Duclair, Sonald Gautam, Archana Ellington, Andrew Prasad, Vinayaka R High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication |
title | High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication |
title_full | High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication |
title_fullStr | High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication |
title_full_unstemmed | High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication |
title_short | High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication |
title_sort | high-affinity rna aptamers against the hiv-1 protease inhibit both in vitro protease activity and late events of viral replication |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345311/ https://www.ncbi.nlm.nih.gov/pubmed/25689224 http://dx.doi.org/10.1038/mtna.2015.1 |
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