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


Zelger-Paulus, Susann; Hadzic, Mélodie C A S; Sigel, Roland K O; Börner, Richard (2020). Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy. In: Arluison, Véronique; Wien, Frank. RNA Spectroscopy. New York: Springer, 1-16.

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.

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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Additional indexing

Item Type:Book Section, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Scopus Subject Areas:Life Sciences > Molecular Biology
Life Sciences > Genetics
Language:English
Date:1 January 2020
Deposited On:16 Apr 2020 07:32
Last Modified:23 Jun 2024 01:39
Publisher:Springer
Series Name:Methods in Molecular Biology
Number:2113
ISSN:1064-3745
ISBN:978-1-0716-0277-5
OA Status:Green
Publisher DOI:https://doi.org/10.1007/978-1-0716-0278-2_1
PubMed ID:32006303
  • Content: Accepted Version
  • Description: Akzeptiere Version ohne Figures