Ribozymes pp 227-251 | Cite as

Single Molecule FRET Characterization of Large Ribozyme Folding

  • Lucia Cardo
  • Krishanthi S. Karunatilaka
  • David Rueda
  • Roland K. O. Sigel
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 848)

Abstract

A procedure to investigate the folding of group II intron by single molecule Fluorescence Resonance Energy Transfer (smFRET) using total internal reflection fluorescence microscopy (TIRFM) is described in this chapter. Using our previous studies on the folding and dynamics of a large ribozyme in the presence of metal ions (i.e., Mg2+ and Ca2+) and/or the DEAD-box protein Mss116 as an example, we here describe step-by-step procedures to perform experiments. smFRET allows the investigation of individual molecules, thus, providing kinetic and mechanistic information hidden in ensemble averaged experiments.

Key words

Single molecule FRET Group II introns Folding TIRF microscopy Dwell times DEAD-box helicases 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Lucia Cardo
    • 1
  • Krishanthi S. Karunatilaka
    • 2
  • David Rueda
    • 2
  • Roland K. O. Sigel
    • 1
  1. 1.Institute of Inorganic ChemistryUniversity of ZurichZurichSwitzerland
  2. 2.Wayne State UniversityDetroitUSA

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