title: Single-molecule pull-down for investigating protein-nucleic acid interactions.
abstract: The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes.
authors.affiliation: Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, The Netherlands.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Fareh
authors.affiliation: Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, The Netherlands.
authors.forename: Luuk
authors.initials: L
authors.lastname: Loeff
authors.affiliation: Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, The Netherlands.
authors.forename: Malwina
authors.initials: M
authors.lastname: Szczepaniak
authors.affiliation: Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, The Netherlands.
authors.forename: Anna C
authors.initials: AC
authors.lastname: Haagsma
authors.affiliation: Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, The Netherlands.
authors.forename: Kyu-Hyeon
authors.initials: KH
authors.lastname: Yeom
authors.affiliation: Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, The Netherlands. Electronic address: c.joo@tudelft.nl.
authors.forename: Chirlmin
authors.initials: C
authors.lastname: Joo
mesh_terms: D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics; D036103:Nanotechnology / Q000379:methods; D009696:Nucleic Acids / Q000737:chemistry / Q000235:genetics; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D009696:Nucleic Acids; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases
keywords: Dicer; Drosha; Protein complex; RNA interference; Single-molecule fluorescence; Single-protein pull-down; TUT4