Visualizing the functional 3D shape and topography of long noncoding RNAs by single-particle atomic force microscopy and in-solution hydrodynamic techniques
| Autor: | Manikandan Karuppasamy, Paolo Annibale, Isabel Chillón, Tina Uroda, Jean-Marie Teulon, Marco Marcia, Jean-Luc Pellequer, Ombeline Pessey |
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| Přispěvatelé: | European Molecular Biology Laboratory [Grenoble] (EMBL), Max Delbrück Center for Molecular Medicine [Berlin] (MDC), Helmholtz-Gemeinschaft = Helmholtz Association, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), ISBG: UMS 3518 CNRS-CEA-UJF-EMBL, BM29-BioSAXS beamline, AFM platform at the IBS, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-15-CE11-0003,lincRNPs,Principes moleculaires de reconnaissance entre la proteine Polycomb SCML2A et longs ARN non-codants(2015), European Project: PJA-20191209284, European Project: ECTZ18552, European Project: 18CN047-00 |
| Rok vydání: | 2019 |
| Předmět: |
MESH: Microscopy
Atomic Force 0303 health sciences [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Structural Biology [q-bio.BM] Atomic force microscopy Chemistry Resolution (electron density) DNA replication RNA Computational biology Microscopy Atomic Force MESH: Image Processing Computer-Assisted General Biochemistry Genetics and Molecular Biology Characterization (materials science) MESH: Hydrodynamics 03 medical and health sciences 0302 clinical medicine MESH: RNA Long Noncoding Microscopy Hydrodynamics Image Processing Computer-Assisted RNA Long Noncoding 030217 neurology & neurosurgery Topology (chemistry) Function (biology) 030304 developmental biology |
| Zdroj: | Nature Protocols Nature Protocols, Nature Publishing Group, 2020, 15 (6), pp.2107-2139. ⟨10.1038/s41596-020-0323-7⟩ Nature Protocols, 2020, 15 (6), pp.2107-2139. ⟨10.1038/s41596-020-0323-7⟩ |
| ISSN: | 1750-2799 |
| DOI: | 10.1038/s41596-020-0323-7⟩ |
| Popis: | International audience; Long noncoding RNAs (lncRNAs) are recently discovered transcripts that regulate vital cellular processes, such as cellular differentiation and DNA replication, and are crucially connected to diseases. Although the 3D structures of lncRNAs are key determinants of their function, the unprecedented molecular complexity of lncRNAs has so far precluded their 3D structural characterization at high resolution. It is thus paramount to develop novel approaches for biochemical and biophysical characterization of these challenging targets. Here, we present a protocol that integrates non-denaturing lncRNA purification with in-solution hydrodynamic analysis and single-particle atomic force microscopy (AFM) imaging to produce highly homogeneous lncRNA preparations and visualize their 3D topology at ~15-Å resolution. Our protocol is suitable for imaging lncRNAs in biologically active conformations and for measuring structural defects of functionally inactive mutants that have been identified by cell-based functional assays. Once optimized for the specific target lncRNA of choice, our protocol leads from cloning to AFM imaging within 3-4 weeks and can be implemented using state-of-the-art biochemical and biophysical instrumentation by trained researchers familiar with RNA handling and supported by AFM and small-angle X-ray scattering (SAXS) experts. |
| Databáze: | OpenAIRE |
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