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Single-Molecule Analysis of RNA Dynamics in Living Cells Using Molecular Beacons

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Epitranscriptomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1870))

Abstract

Over the past decade, emerging evidence has indicated that long intergenic noncoding RNAs (lincRNAs), a class of RNA transcripts greater than 200 nt in length, function as key regulators of gene expression in cellular physiology and pathogenesis. Greater understanding of lincRNA activities, particularly in the context of subcellular localization and dynamic regulation at the single-molecule level, is expected to provide in-depth understanding of molecular mechanisms that regulate cell behavior and disease evolution. We have recently developed a fluorescence-imaging approach to investigate RNA dynamics in living cells at the single-molecule level. This approach entails the use of molecular beacons (MBs), which are a class of stem-loop forming oligonculeotide-based probes that emit detectable fluorescence upon binding to target sequence, and tandem repeats of MB target sequences integrated to the target RNA sequence. Binding of the MBs to the tandem repeats could illuminate the target RNA as a bright spot when imaged by conventional fluorescence microscopy, making the MB-based imaging approach a versatile tool for RNA analysis across laboratories. In this chapter, we describe the development of the MB-based approach and its application for imaging single NEAT1 lincRNA transcripts in living cells.

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Acknowledgments

This project was supported by grants from the National Key R&D Program of China (Nos. 2016YFA0501603 and 2016YFA0100702), the National Natural Science Foundation of China (Nos. 31771583 and 81371613), the Beijing Natural Science Foundation (7162114) and China’s 1000 Young Talent Award program.

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Authors and Affiliations

  1. Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China

    Mingming Chen, Shiqi Mao, Xiaotian Wu, Zhao Ma, Yantao Yang, Christopher J. Krueger & Antony K. Chen

  2. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Mingming Chen

  3. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Mingming Chen

  4. Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA

    Christopher J. Krueger

Authors
  1. Mingming Chen
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  2. Shiqi Mao
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  3. Xiaotian Wu
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  4. Zhao Ma
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  5. Yantao Yang
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  6. Christopher J. Krueger
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  7. Antony K. Chen
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Corresponding author

Correspondence to Antony K. Chen .

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Editors and Affiliations

  1. Department of Pathology, Yale University School of Medicine, New Haven, CT, USA

    Narendra Wajapeyee

  2. Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Romi Gupta

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Chen, M. et al. (2019). Single-Molecule Analysis of RNA Dynamics in Living Cells Using Molecular Beacons. In: Wajapeyee, N., Gupta, R. (eds) Epitranscriptomics. Methods in Molecular Biology, vol 1870. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8808-2_2

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  • DOI: https://doi.org/10.1007/978-1-4939-8808-2_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8807-5

  • Online ISBN: 978-1-4939-8808-2

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