24. Studying RNA Using Single Molecule Fluorescence Resonance Energy Transfer

  1. Roland K. Hartmann3,
  2. Albrecht Bindereif4,
  3. Astrid Schön5 and
  4. Eric Westhof6
  1. Felix Spenkuch1,
  2. Olwen Domingo1,
  3. Gerald Hinze2,
  4. Thomas Basché2 and
  5. Mark Helm1

Published Online: 28 MAR 2014

DOI: 10.1002/9783527647064.ch24

Handbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition

Handbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition

How to Cite

Spenkuch, F., Domingo, O., Hinze, G., Basché, T. and Helm, M. (2014) Studying RNA Using Single Molecule Fluorescence Resonance Energy Transfer, in Handbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition (eds R. K. Hartmann, A. Bindereif, A. Schön and E. Westhof), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527647064.ch24

Editor Information

  1. 3

    Philipps-Universität Marburg, Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Marbacher Weg 6, Building C, 35037 Marburg, Germany

  2. 4

    Justus-Liebig-Universität Gießen, Fachbereich Biologie und Chemie, Institut für Biochemie, Heinrich-Buff-Ring 58, 35392 Gießen, Germany

  3. 5

    Universität Leipzig, Molekulare Zelltherapie, Biotechnologisch-Biomedizinisches Zentrum, Deutscher Platz 5, 04105 Leipzig, Germany

  4. 6

    Université de Strasbourg, Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et, Cellulaire du CNRS, 67084 Strasbourg, France

Author Information

  1. 1

    Institute of Pharmacy and Biochemistry, Johannes Gutenberg-Universität, Germany

  2. 2

    Institute of Physical Chemistry, Johannes Gutenberg-Universität, Duesbergweg 10-14, 55099 Mainz, Germany

Publication History

  1. Published Online: 28 MAR 2014
  2. Published Print: 9 APR 2014

ISBN Information

Print ISBN: 9783527327645

Online ISBN: 9783527647064



  • Fluorescence resonance energy transfer (FRET);
  • Förster radius;
  • single molecule FRET (smFRET);
  • donor fluorophore;
  • acceptor fluorophore;
  • FRET dye pairs;
  • emission spectra;
  • excitation spectra;
  • photobleaching;
  • antifading agents;
  • bulk FRET measurements;
  • dye labeling of RNA by click chemistry;
  • measurement chamber design;
  • immobilization of RNA by biotin-streptavidin interaction;
  • RNA structure, conformation, dynamics, and structural homogeneity;
  • PEG-coating of surfaces


The intrinsic structure of RNA and in particular the dynamics of the same are of paramount importance for RNA function. Recent work, e.g., on riboswitches and ribosomes, shows the vast complexity of these dynamics. Consequently the interest in biochemical and biophysical methods to investigate these dynamics is rising. Here we describe the use of single molecule fluorescence resonance energy transfer (FRET) to investigate structural dynamics of individual immobilized RNA molecules. Such observations on the single molecule level allow the description of equilibria between structural populations, even down to transient subpopulations, which would otherwise been hidden in the ensemble average. This chapter starts with a brief introduction into FRET, followed by considerations on construct design, as well as dye choice and the layout of an instrumental setup used in our group. In an experimental part, RNA labeling methods are presented, as well as measurement chamber assembly, buffers that enhance dye performance, and actual sample preparation.