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Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs

The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical mod...

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Autores principales: Czapik, Tomasz, Piasecka, Julita, Kierzek, Ryszard, Kierzek, Elzbieta
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9217987/
https://www.ncbi.nlm.nih.gov/pubmed/35784013
http://dx.doi.org/10.1016/j.omtn.2022.05.035
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author Czapik, Tomasz
Piasecka, Julita
Kierzek, Ryszard
Kierzek, Elzbieta
author_facet Czapik, Tomasz
Piasecka, Julita
Kierzek, Ryszard
Kierzek, Elzbieta
author_sort Czapik, Tomasz
collection PubMed
description The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical modifications aiming to improve the RNA cleavage activity of ribozymes. We introduced an additional hairpin motif and attempted to select ribozyme-target pairs with sequence features that enable the potential formation of the trans-Hoogsteen interactions that are present in full-length, highly active hammerhead ribozymes. We placed structurally defined guanosine analogs into the ribozyme catalytic core. Herein, the significantly improved synthesis of 2′-deoxy-2′-fluoroarabinoguanosine derivatives is described. The most potent hammerhead ribozymes were applied to chimeric short hairpin RNA (shRNA)-ribozyme plasmid constructs to improve the antiviral activity of the two components. The modified hammerhead ribozymes showed moderate cleavage activity. Treatment of IAV-infected Madin-Darby canine kidney (MDCK) cells with the plasmid constructs resulted in significant inhibition of virus replication. Real-time PCR analysis revealed a significant (80%–88%) reduction in viral RNA when plasmids carriers were used. A focus formation assay (FFA) for chimeric plasmids showed inhibition of virus replication by 1.6–1.7 log(10) units, whereas the use of plasmids carrying ribozymes or shRNAs alone resulted in lower inhibition.
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spelling pubmed-92179872022-06-30 Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs Czapik, Tomasz Piasecka, Julita Kierzek, Ryszard Kierzek, Elzbieta Mol Ther Nucleic Acids Original Article The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical modifications aiming to improve the RNA cleavage activity of ribozymes. We introduced an additional hairpin motif and attempted to select ribozyme-target pairs with sequence features that enable the potential formation of the trans-Hoogsteen interactions that are present in full-length, highly active hammerhead ribozymes. We placed structurally defined guanosine analogs into the ribozyme catalytic core. Herein, the significantly improved synthesis of 2′-deoxy-2′-fluoroarabinoguanosine derivatives is described. The most potent hammerhead ribozymes were applied to chimeric short hairpin RNA (shRNA)-ribozyme plasmid constructs to improve the antiviral activity of the two components. The modified hammerhead ribozymes showed moderate cleavage activity. Treatment of IAV-infected Madin-Darby canine kidney (MDCK) cells with the plasmid constructs resulted in significant inhibition of virus replication. Real-time PCR analysis revealed a significant (80%–88%) reduction in viral RNA when plasmids carriers were used. A focus formation assay (FFA) for chimeric plasmids showed inhibition of virus replication by 1.6–1.7 log(10) units, whereas the use of plasmids carrying ribozymes or shRNAs alone resulted in lower inhibition. American Society of Gene & Cell Therapy 2022-05-31 /pmc/articles/PMC9217987/ /pubmed/35784013 http://dx.doi.org/10.1016/j.omtn.2022.05.035 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Original Article
Czapik, Tomasz
Piasecka, Julita
Kierzek, Ryszard
Kierzek, Elzbieta
Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs
title Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs
title_full Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs
title_fullStr Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs
title_full_unstemmed Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs
title_short Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs
title_sort structural variants and modifications of hammerhead ribozymes targeting influenza a virus conserved structural motifs
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9217987/
https://www.ncbi.nlm.nih.gov/pubmed/35784013
http://dx.doi.org/10.1016/j.omtn.2022.05.035
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