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Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication

Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtain...

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Autores principales: Knizhnik, Ekaterina, Chumakov, Stepan, Svetlova, Julia, Pavlova, Iulia, Khodarovich, Yuri, Brylev, Vladimir, Severov, Vjacheslav, Alieva, Rugiya, Kozlovskaya, Liubov, Andreev, Dmitry, Aralov, Andrey, Varizhuk, Anna
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10668963/
https://www.ncbi.nlm.nih.gov/pubmed/38002341
http://dx.doi.org/10.3390/biom13111660
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author Knizhnik, Ekaterina
Chumakov, Stepan
Svetlova, Julia
Pavlova, Iulia
Khodarovich, Yuri
Brylev, Vladimir
Severov, Vjacheslav
Alieva, Rugiya
Kozlovskaya, Liubov
Andreev, Dmitry
Aralov, Andrey
Varizhuk, Anna
author_facet Knizhnik, Ekaterina
Chumakov, Stepan
Svetlova, Julia
Pavlova, Iulia
Khodarovich, Yuri
Brylev, Vladimir
Severov, Vjacheslav
Alieva, Rugiya
Kozlovskaya, Liubov
Andreev, Dmitry
Aralov, Andrey
Varizhuk, Anna
author_sort Knizhnik, Ekaterina
collection PubMed
description Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA–DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals.
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spelling pubmed-106689632023-11-17 Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication Knizhnik, Ekaterina Chumakov, Stepan Svetlova, Julia Pavlova, Iulia Khodarovich, Yuri Brylev, Vladimir Severov, Vjacheslav Alieva, Rugiya Kozlovskaya, Liubov Andreev, Dmitry Aralov, Andrey Varizhuk, Anna Biomolecules Article Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA–DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals. MDPI 2023-11-17 /pmc/articles/PMC10668963/ /pubmed/38002341 http://dx.doi.org/10.3390/biom13111660 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Knizhnik, Ekaterina
Chumakov, Stepan
Svetlova, Julia
Pavlova, Iulia
Khodarovich, Yuri
Brylev, Vladimir
Severov, Vjacheslav
Alieva, Rugiya
Kozlovskaya, Liubov
Andreev, Dmitry
Aralov, Andrey
Varizhuk, Anna
Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication
title Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication
title_full Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication
title_fullStr Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication
title_full_unstemmed Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication
title_short Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication
title_sort unwinding the sars-cov-2 ribosomal frameshifting pseudoknot with lna and g-clamp-modified phosphorothioate oligonucleotides inhibits viral replication
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10668963/
https://www.ncbi.nlm.nih.gov/pubmed/38002341
http://dx.doi.org/10.3390/biom13111660
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