Single molecule FRET characterization of large ribozyme folding

Methods Mol Biol. 2012:848:227-51. doi: 10.1007/978-1-61779-545-9_15.

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., Mg(2+) and Ca(2+)) 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Calibration
  • DEAD-box RNA Helicases / metabolism
  • Fluorescence Polarization
  • Fluorescence Resonance Energy Transfer / instrumentation
  • Fluorescence Resonance Energy Transfer / methods*
  • Introns / genetics
  • Magnesium Chloride / pharmacology
  • Microfluidic Analytical Techniques
  • Molecular Sequence Data
  • Nucleic Acid Conformation* / drug effects
  • Polyethylene Glycols / chemistry
  • RNA, Catalytic / chemistry*
  • RNA, Catalytic / genetics
  • RNA, Catalytic / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae Proteins / metabolism
  • Transcription, Genetic / drug effects

Substances

  • RNA, Catalytic
  • Saccharomyces cerevisiae Proteins
  • Magnesium Chloride
  • Polyethylene Glycols
  • MSS116 protein, S cerevisiae
  • DEAD-box RNA Helicases