Single molecule FRET characterization of large ribozyme folding.
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.
Institute of Inorganic Chemistry, University of Zurich, Zurich, Switzerland.
Lucia
L
Cardo
Krishanthi S
KS
Karunatilaka
David
D
Rueda
Roland K O
RK
Sigel
D001483:Base Sequence; D002138:Calibration; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D007438:Introns / Q000235:genetics; D015636:Magnesium Chloride / Q000494:pharmacology; D046210:Microfluidic Analytical Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011092:Polyethylene Glycols / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D016337:RNA, Catalytic; D029701:Saccharomyces cerevisiae Proteins; D015636:Magnesium Chloride; D011092:Polyethylene Glycols; C058150:MSS116 protein, S cerevisiae; D053487:DEAD-box RNA Helicases