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Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling
Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells. Our recently reported RNA-TAG (transglycosylation at guanine) approach uses an RNA-modifying enzyme, tRNA-guanine transglycosylase (TGT), t...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Royal Society of Chemistry
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635419/ https://www.ncbi.nlm.nih.gov/pubmed/29081948 http://dx.doi.org/10.1039/c7sc03150e |
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author | Zhou, Cun Yu Alexander, Seth C. Devaraj, Neal K. |
author_facet | Zhou, Cun Yu Alexander, Seth C. Devaraj, Neal K. |
author_sort | Zhou, Cun Yu |
collection | PubMed |
description | Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells. Our recently reported RNA-TAG (transglycosylation at guanine) approach uses an RNA-modifying enzyme, tRNA-guanine transglycosylase (TGT), to accomplish covalent labeling of an RNA of interest with fluorescent tracking agents in a highly selective and efficient manner. Unfortunately, labeling by this method currently suffers from a high nonspecific fluorescent background and is currently unsuitable for imaging RNA within complex cellular environments. Herein we report the design and synthesis of novel fluorogenic thiazole orange probes that significantly lower nonspecific binding and background fluorescence and, as a result, provide up to a 100-fold fluorescence intensity increase after labeling. Using these fluorogenic labeling agents, we were able to image mRNA expressed in Chinese Hamster Ovary cells in a wash-free manner. |
format | Online Article Text |
id | pubmed-5635419 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Royal Society of Chemistry |
record_format | MEDLINE/PubMed |
spelling | pubmed-56354192017-10-27 Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling Zhou, Cun Yu Alexander, Seth C. Devaraj, Neal K. Chem Sci Chemistry Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells. Our recently reported RNA-TAG (transglycosylation at guanine) approach uses an RNA-modifying enzyme, tRNA-guanine transglycosylase (TGT), to accomplish covalent labeling of an RNA of interest with fluorescent tracking agents in a highly selective and efficient manner. Unfortunately, labeling by this method currently suffers from a high nonspecific fluorescent background and is currently unsuitable for imaging RNA within complex cellular environments. Herein we report the design and synthesis of novel fluorogenic thiazole orange probes that significantly lower nonspecific binding and background fluorescence and, as a result, provide up to a 100-fold fluorescence intensity increase after labeling. Using these fluorogenic labeling agents, we were able to image mRNA expressed in Chinese Hamster Ovary cells in a wash-free manner. Royal Society of Chemistry 2017-10-01 2017-08-29 /pmc/articles/PMC5635419/ /pubmed/29081948 http://dx.doi.org/10.1039/c7sc03150e Text en This journal is © The Royal Society of Chemistry 2017 http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Chemistry Zhou, Cun Yu Alexander, Seth C. Devaraj, Neal K. Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling |
title | Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling
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title_full | Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling
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title_fullStr | Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling
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title_full_unstemmed | Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling
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title_short | Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling
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title_sort | fluorescent turn-on probes for wash-free mrna imaging via covalent site-specific enzymatic labeling |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635419/ https://www.ncbi.nlm.nih.gov/pubmed/29081948 http://dx.doi.org/10.1039/c7sc03150e |
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