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Optimizing Molecular Beacons for Intracellular Analysis of RNA

  • Protocol
RNA Detection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1649))

Abstract

Conventional molecular beacons (MBs) have been used extensively for imaging specific endogenous RNAs in living cells, but their tendency to generate false-positive signals as a result of nuclease degradation and/or nonspecific binding limits sensitive and accurate imaging of intracellular RNAs. In an attempt to overcome this limitation, MBs have been synthesized with various chemically modified oligonucleotide backbones to confer greater biostability. We have recently developed a new MB architecture composed of 2′-O-methyl RNA (2Me), a fully phosphorothioate (PS) modified loop domain and a phosphodiester stem (2Me/PSLOOP MB). We showed that this new MB exhibits a marginal level of false-positive signals and enables accurate single-molecule imaging of target RNA in living cells. In this chapter, we describe detailed methods that led us to conclude that, among various PS-modified configurations, the 2Me/PSLOOP MB is an optimal design for intracellular RNA analysis.

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Acknowledgments

This project was supported by grants from the National Basic Research Program of China (2016YFA0100702), the National Natural Science Foundation of China (81371613), and the Beijing Natural Science Foundation (7162114).

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Authors and Affiliations

  1. Department of Biomedical Engineering, College of Engineering, Peking University, No. 5 Yiheyuan Rd. Haidian District, Beijing, 100871, China

    Mingming Chen, Yantao Yang, Christopher J. Krueger & Antony K. Chen

  2. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China

    Mingming Chen

  3. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China

    Mingming Chen

  4. Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA

    Christopher J. Krueger

Authors
  1. Mingming Chen
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  2. Yantao Yang
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  3. Christopher J. Krueger
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  4. Antony K. Chen
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Corresponding author

Correspondence to Antony K. Chen .

Editor information

Editors and Affiliations

  1. Developmental Biology Unit, EMBL Heidelberg, Heidelberg, Germany

    Imre Gaspar

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Chen, M., Yang, Y., Krueger, C.J., Chen, A.K. (2018). Optimizing Molecular Beacons for Intracellular Analysis of RNA. In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_16

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  • DOI: https://doi.org/10.1007/978-1-4939-7213-5_16

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7212-8

  • Online ISBN: 978-1-4939-7213-5

  • eBook Packages: Springer Protocols

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