title: Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.
abstract: TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform.
authors.affiliation: Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Hye Ran
authors.initials: HR
authors.lastname: Koh
authors.affiliation: Department of Bioengineering, University of Illinois, Urbana, IL, USA.
authors.forename: Xinlei
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA. Electronic address: smyong@jhu.edu.
authors.forename: Sua
authors.initials: S
authors.lastname: Myong
mesh_terms: D000818:Animals; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008322:Mammals; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins
keywords: Diffusion activity of dsRBP; Double-strand RNA binding protein (dsRBP); Single molecule FRET (smFRET); Single molecule PIFE (smPIFE); Single molecule pull down (SiMPull); TAR RNA binding protein (TRBP); Three color FRET