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Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy

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RNA Spectroscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2113))

Abstract

Imaging fluorescently labeled biomolecules on a single-molecule level is a well-established technique to follow intra- and intermolecular processes in time, usually hidden in the ensemble average. The classical approach comprises surface immobilization of the molecule of interest, which increases the risk of restricting the natural behavior due to surface interactions. Encapsulation of such biomolecules into surface-tethered phospholipid vesicles enables to follow one molecule at a time, freely diffusing and without disturbing surface interactions. Further, the encapsulation allows to keep reaction partners (reactants and products) in close proximity and enables higher temperatures otherwise leading to desorption of the direct immobilized biomolecules.

Here, we describe a detailed protocol for the encapsulation of a catalytically active RNA starting from surface passivation over RNA encapsulation to data evaluation of single-molecule FRET experiments in TIRF microscopy. We present an optimized procedure that preserves RNA functionality and applies to investigations of, e.g., large ribozymes and RNAs, where direct immobilization is structurally not possible.

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Acknowledgments

Financial support from the Swiss National Science Foundation [to RKOS], the UZH Forschungskredit [FK-13-095, FK-15-09 to SZP, FK-13-091 to MCASH, FK-14-096, FK-15-095 to RB], the UZH Stiftung für wissenschaftliche Forschung [to RKOS and RB], and the University of Zurich is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Chemistry, University of Zurich, Zurich, Switzerland

    Susann Zelger-Paulus, Mélodie C. A. S. Hadzic, Roland K. O. Sigel & Richard Börner

  2. Laserinstitut Hochschule Mittweida, University of Applied Sciences Mittweida, Mittweida, Germany

    Richard Börner

Authors
  1. Susann Zelger-Paulus
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  2. Mélodie C. A. S. Hadzic
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  3. Roland K. O. Sigel
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  4. Richard Börner
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Corresponding authors

Correspondence to Roland K. O. Sigel or Richard Börner .

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Editors and Affiliations

  1. Université de Paris, Paris, France, Laboratoire Léon Brillouin LLB, CEA, CNRS UMR12, Université Paris Saclay, Gif-sur-Yvette, France

    Véronique Arluison

  2. Synchrotron SOLEIL L’Orme des Merisiers Saint Aubin, Gif-sur-Yvette, France

    Frank Wien

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Zelger-Paulus, S., Hadzic, M.C.A.S., Sigel, R.K.O., Börner, R. (2020). Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy. In: Arluison, V., Wien, F. (eds) RNA Spectroscopy. Methods in Molecular Biology, vol 2113. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0278-2_1

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  • DOI: https://doi.org/10.1007/978-1-0716-0278-2_1

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