Volume 848 of the series Methods in Molecular Biology pp 227-251


Single Molecule FRET Characterization of Large Ribozyme Folding

  • Lucia CardoAffiliated withInstitute of Inorganic Chemistry, University of Zurich
  • , Krishanthi S. KarunatilakaAffiliated withWayne State University
  • , David RuedaAffiliated withWayne State University Email author 
  • , Roland K. O. SigelAffiliated withInstitute of Inorganic Chemistry, University of Zurich

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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