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Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA
The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology lab...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10209184/ https://www.ncbi.nlm.nih.gov/pubmed/37225690 http://dx.doi.org/10.1038/s41467-023-38579-2 |
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author | Liang, Yufeng Willey, Sydney Chung, Yu-Chieh Lo, Yi-Meng Miao, Shiqin Rundell, Sarah Tu, Li-Chun Bong, Dennis |
author_facet | Liang, Yufeng Willey, Sydney Chung, Yu-Chieh Lo, Yi-Meng Miao, Shiqin Rundell, Sarah Tu, Li-Chun Bong, Dennis |
author_sort | Liang, Yufeng |
collection | PubMed |
description | The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology labs, the protein labels add significant mass to the bound RNA, which potentially impacts steric accessibility and native RNA biology. We have previously demonstrated that internal, genetically encoded, uridine-rich internal loops (URILs) comprised of four contiguous UU pairs (8 nt) in RNA may be targeted with minimal structural perturbation by triplex hybridization with 1 kD bifacial peptide nucleic acids (bPNAs). A URIL-targeting strategy for RNA and DNA tracking would avoid the use of cumbersome protein fusion labels and minimize structural alterations to the RNA of interest. Here we show that URIL-targeting fluorogenic bPNA probes in cell media can penetrate cell membranes and effectively label RNAs and RNPs in fixed and live cells. This method, which we call fluorogenic U-rich internal loop (FLURIL) tagging, was internally validated through the use of RNAs bearing both URIL and MS2 labeling sites. Notably, a direct comparison of CRISPR-dCas labeled genomic loci in live U2OS cells revealed that FLURIL-tagged gRNA yielded loci with signal to background up to 7X greater than loci targeted by guide RNA modified with an array of eight MS2 hairpins. Together, these data show that FLURIL tagging provides a versatile scope of intracellular RNA and DNA tracking while maintaining a light molecular footprint and compatibility with existing methods. |
format | Online Article Text |
id | pubmed-10209184 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-102091842023-05-26 Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA Liang, Yufeng Willey, Sydney Chung, Yu-Chieh Lo, Yi-Meng Miao, Shiqin Rundell, Sarah Tu, Li-Chun Bong, Dennis Nat Commun Article The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology labs, the protein labels add significant mass to the bound RNA, which potentially impacts steric accessibility and native RNA biology. We have previously demonstrated that internal, genetically encoded, uridine-rich internal loops (URILs) comprised of four contiguous UU pairs (8 nt) in RNA may be targeted with minimal structural perturbation by triplex hybridization with 1 kD bifacial peptide nucleic acids (bPNAs). A URIL-targeting strategy for RNA and DNA tracking would avoid the use of cumbersome protein fusion labels and minimize structural alterations to the RNA of interest. Here we show that URIL-targeting fluorogenic bPNA probes in cell media can penetrate cell membranes and effectively label RNAs and RNPs in fixed and live cells. This method, which we call fluorogenic U-rich internal loop (FLURIL) tagging, was internally validated through the use of RNAs bearing both URIL and MS2 labeling sites. Notably, a direct comparison of CRISPR-dCas labeled genomic loci in live U2OS cells revealed that FLURIL-tagged gRNA yielded loci with signal to background up to 7X greater than loci targeted by guide RNA modified with an array of eight MS2 hairpins. Together, these data show that FLURIL tagging provides a versatile scope of intracellular RNA and DNA tracking while maintaining a light molecular footprint and compatibility with existing methods. Nature Publishing Group UK 2023-05-24 /pmc/articles/PMC10209184/ /pubmed/37225690 http://dx.doi.org/10.1038/s41467-023-38579-2 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Liang, Yufeng Willey, Sydney Chung, Yu-Chieh Lo, Yi-Meng Miao, Shiqin Rundell, Sarah Tu, Li-Chun Bong, Dennis Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA |
title | Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA |
title_full | Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA |
title_fullStr | Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA |
title_full_unstemmed | Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA |
title_short | Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA |
title_sort | intracellular rna and dna tracking by uridine-rich internal loop tagging with fluorogenic bpna |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10209184/ https://www.ncbi.nlm.nih.gov/pubmed/37225690 http://dx.doi.org/10.1038/s41467-023-38579-2 |
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