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Single-Molecule FRET Studies of RNA Structural Rearrangements and RNA-RNA Interactions

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Riboregulator Design and Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2518))

Abstract

RNA-guided regulation of gene expression is found in all cell types. In this mode of regulation, antisense interactions between the regulatory RNA and its target are typically facilitated by a protein partner. Single-molecule fluorescence microscopy is a powerful tool for dissecting the conformational states and intermediates that contribute to target recognition. This chapter describes protocols for studying target recognition by bacterial small RNAs and their chaperone Hfq on the single-molecule level, using a total internal reflection fluorescence microscope. The sections cover the design of suitable RNA substrates for sRNA-mRNA annealing reactions, preparation of internally labeled mRNA for detecting conformational changes in the target, and key steps of the data analysis. These protocols can be adapted to other RNA-binding proteins that chaperone RNA interactions.

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Acknowledgments

This work was supported by the National Institutes of Health (R35GM136351 to S.A.W.). The authors thank Subrata Panja and Andrew Santiago-Frangos for contributing to early versions of these protocols.

Author information

Authors and Affiliations

  1. T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA

    Ewelina M. Małecka & Sarah A. Woodson

  2. Department of Molecular Biology and Genetics, Johns Hopkins Medical School, Baltimore, MD, USA

    Boyang Hua

Authors
  1. Ewelina M. Małecka
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  2. Boyang Hua
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  3. Sarah A. Woodson
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Corresponding author

Correspondence to Sarah A. Woodson .

Editor information

Editors and Affiliations

  1. Biosciences Department, Rice University, Houston, TX, USA

    James Chappell

  2. Department of Biology, California State University, Northridge, Northridge, CA, USA

    Melissa K. Takahashi

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Małecka, E.M., Hua, B., Woodson, S.A. (2022). Single-Molecule FRET Studies of RNA Structural Rearrangements and RNA-RNA Interactions. In: Chappell, J., Takahashi, M.K. (eds) Riboregulator Design and Analysis. Methods in Molecular Biology, vol 2518. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2421-0_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2421-0_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2420-3

  • Online ISBN: 978-1-0716-2421-0

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