A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level

Abstract Analysis of RNA dynamics and localization at the single-molecule level in living cells has been predominantly achieved by engineering target RNAs with large insertions of tandem repeat sequences that are bound by protein-based or oligonucleotide-based fluorescent probes. Thus, individual RN...

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Autores principales:	Mingming Chen, Zhao Ma, Xiaotian Wu, Shiqi Mao, Yantao Yang, Jie Tan, Christopher J. Krueger, Antony K. Chen
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/830019b1912f4a87a66e26d327bea6da
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spelling	oai:doaj.org-article:830019b1912f4a87a66e26d327bea6da2021-12-02T15:18:51ZA molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level10.1038/s41598-017-01740-12045-2322https://doaj.org/article/830019b1912f4a87a66e26d327bea6da2017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-01740-1https://doaj.org/toc/2045-2322Abstract Analysis of RNA dynamics and localization at the single-molecule level in living cells has been predominantly achieved by engineering target RNAs with large insertions of tandem repeat sequences that are bound by protein-based or oligonucleotide-based fluorescent probes. Thus, individual RNAs are tagged by multiple fluorescent probes, making them detectable by fluorescence microscopy. Since large insertions may affect RNA processes including trafficking and localization, here we present a strategy to visualize single RNA transcripts in living cells using molecular beacons (MBs) - fluorogenic oligonucleotide probes - with minimal target engineering. The MBs are composed of 2′-O-methyl RNAs with a fully phosphorothioate-modified loop domain (2Me/PSLOOP MBs), an architecture that elicits marginal levels of nonspecific signals in cells. We showed that MBs can detect single transcripts containing as few as 8 target repeat sequences with ~90% accuracy. In both the nucleus and the cytoplasm, mRNAs harboring 8 repeats moved faster than those with 32 repeats, suggesting that intracellular activities are less impeded by smaller engineered insertions. We then report the first MB-based imaging of intracellular dynamics and localization of single long noncoding RNAs (lncRNAs). We envision the proposed minimally-engineered, MB-based technology for live-cell single-molecule RNA imaging could facilitate new discoveries in RNA research.Mingming ChenZhao MaXiaotian WuShiqi MaoYantao YangJie TanChristopher J. KruegerAntony K. ChenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Mingming Chen Zhao Ma Xiaotian Wu Shiqi Mao Yantao Yang Jie Tan Christopher J. Krueger Antony K. Chen A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level
description	Abstract Analysis of RNA dynamics and localization at the single-molecule level in living cells has been predominantly achieved by engineering target RNAs with large insertions of tandem repeat sequences that are bound by protein-based or oligonucleotide-based fluorescent probes. Thus, individual RNAs are tagged by multiple fluorescent probes, making them detectable by fluorescence microscopy. Since large insertions may affect RNA processes including trafficking and localization, here we present a strategy to visualize single RNA transcripts in living cells using molecular beacons (MBs) - fluorogenic oligonucleotide probes - with minimal target engineering. The MBs are composed of 2′-O-methyl RNAs with a fully phosphorothioate-modified loop domain (2Me/PSLOOP MBs), an architecture that elicits marginal levels of nonspecific signals in cells. We showed that MBs can detect single transcripts containing as few as 8 target repeat sequences with ~90% accuracy. In both the nucleus and the cytoplasm, mRNAs harboring 8 repeats moved faster than those with 32 repeats, suggesting that intracellular activities are less impeded by smaller engineered insertions. We then report the first MB-based imaging of intracellular dynamics and localization of single long noncoding RNAs (lncRNAs). We envision the proposed minimally-engineered, MB-based technology for live-cell single-molecule RNA imaging could facilitate new discoveries in RNA research.
format	article
author	Mingming Chen Zhao Ma Xiaotian Wu Shiqi Mao Yantao Yang Jie Tan Christopher J. Krueger Antony K. Chen
author_facet	Mingming Chen Zhao Ma Xiaotian Wu Shiqi Mao Yantao Yang Jie Tan Christopher J. Krueger Antony K. Chen
author_sort	Mingming Chen
title	A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level
title_short	A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level
title_full	A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level
title_fullStr	A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level
title_full_unstemmed	A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level
title_sort	molecular beacon-based approach for live-cell imaging of rna transcripts with minimal target engineering at the single-molecule level
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/830019b1912f4a87a66e26d327bea6da
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A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level

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