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Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5’ untranslated region

One of the hallmarks of RNA viruses is highly structured untranslated regions (UTRs) in their genomes. These conserved RNA structures are often essential for viral replication, transcription, or translation. In this report, we discovered and optimized a new type of coumarin derivatives, such as C30...

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Autores principales: Tang, Zhichao, Hegde, Shalakha, Hao, Siyuan, Selvaraju, Manikandan, Qiu, Jianming, Wang, Jingxin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10103992/
https://www.ncbi.nlm.nih.gov/pubmed/37066172
http://dx.doi.org/10.1101/2023.04.03.535453
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author Tang, Zhichao
Hegde, Shalakha
Hao, Siyuan
Selvaraju, Manikandan
Qiu, Jianming
Wang, Jingxin
author_facet Tang, Zhichao
Hegde, Shalakha
Hao, Siyuan
Selvaraju, Manikandan
Qiu, Jianming
Wang, Jingxin
author_sort Tang, Zhichao
collection PubMed
description One of the hallmarks of RNA viruses is highly structured untranslated regions (UTRs) in their genomes. These conserved RNA structures are often essential for viral replication, transcription, or translation. In this report, we discovered and optimized a new type of coumarin derivatives, such as C30 and C34, which bind to a four-way RNA helix called SL5 in the 5’ UTR of the SARS-CoV-2 RNA genome. To locate the binding site, we developed a novel sequencing-based method namely cgSHAPE-seq, in which the acylating chemical probe was directed to crosslink with the 2’-OH groups of ribose at the ligand binding site. This crosslinked RNA could then create read-through mutations during reverse transcription (i.e., primer extension) at single-nucleotide resolution to uncover the acylation locations. cgSHAPE-seq unambiguously determined that a bulged G in SL5 was the primary binding site of C30 in the SARS-CoV-2 5’ UTR, which was validated through mutagenesis and in vitro binding experiments. C30 was further used as a warhead in RNA-degrading chimeras to reduce viral RNA expression levels. We demonstrated that replacing the acylating moiety in the cgSHAPE probe with ribonuclease L recruiter (RLR) moieties yielded RNA degraders active in the in vitro RNase L degradation assay and SARS-CoV-2 5’ UTR expressing cells. We further explored another RLR conjugation site on the E ring of C30/C34 and discovered improved RNA degradation activities in vitro and in cells. The optimized RNA-degrading chimera C64 inhibited live virus replication in lung epithelial carcinoma cells.
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spelling pubmed-101039922023-04-15 Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5’ untranslated region Tang, Zhichao Hegde, Shalakha Hao, Siyuan Selvaraju, Manikandan Qiu, Jianming Wang, Jingxin bioRxiv Article One of the hallmarks of RNA viruses is highly structured untranslated regions (UTRs) in their genomes. These conserved RNA structures are often essential for viral replication, transcription, or translation. In this report, we discovered and optimized a new type of coumarin derivatives, such as C30 and C34, which bind to a four-way RNA helix called SL5 in the 5’ UTR of the SARS-CoV-2 RNA genome. To locate the binding site, we developed a novel sequencing-based method namely cgSHAPE-seq, in which the acylating chemical probe was directed to crosslink with the 2’-OH groups of ribose at the ligand binding site. This crosslinked RNA could then create read-through mutations during reverse transcription (i.e., primer extension) at single-nucleotide resolution to uncover the acylation locations. cgSHAPE-seq unambiguously determined that a bulged G in SL5 was the primary binding site of C30 in the SARS-CoV-2 5’ UTR, which was validated through mutagenesis and in vitro binding experiments. C30 was further used as a warhead in RNA-degrading chimeras to reduce viral RNA expression levels. We demonstrated that replacing the acylating moiety in the cgSHAPE probe with ribonuclease L recruiter (RLR) moieties yielded RNA degraders active in the in vitro RNase L degradation assay and SARS-CoV-2 5’ UTR expressing cells. We further explored another RLR conjugation site on the E ring of C30/C34 and discovered improved RNA degradation activities in vitro and in cells. The optimized RNA-degrading chimera C64 inhibited live virus replication in lung epithelial carcinoma cells. Cold Spring Harbor Laboratory 2023-10-23 /pmc/articles/PMC10103992/ /pubmed/37066172 http://dx.doi.org/10.1101/2023.04.03.535453 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Tang, Zhichao
Hegde, Shalakha
Hao, Siyuan
Selvaraju, Manikandan
Qiu, Jianming
Wang, Jingxin
Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5’ untranslated region
title Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5’ untranslated region
title_full Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5’ untranslated region
title_fullStr Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5’ untranslated region
title_full_unstemmed Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5’ untranslated region
title_short Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5’ untranslated region
title_sort chemical-guided shape sequencing (cgshape-seq) informs the binding site of rna-degrading chimeras targeting sars-cov-2 5’ untranslated region
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10103992/
https://www.ncbi.nlm.nih.gov/pubmed/37066172
http://dx.doi.org/10.1101/2023.04.03.535453
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