Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer‐Protein Interactions (RFAP) in Live Bacterial Cells

Maria Valencia‐Burton1, Natalia E. Broude1

1 Center for Advanced Biotechnology, College of Engineering, Department of Biomedical Engineering, Boston University, Boston
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.11
DOI:  10.1002/0471143030.cb1711s37
Online Posting Date:  December, 2007
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Abstract

This unit describes a method allowing RNA visualization in live cells. The method is based on fluorescent protein complementation regulated by RNA‐aptamer/RNA‐binding protein interactions. Based on these two principles, a fluorescent ribonucleoprotein complex is assembled inside the cell only in response to the presence of the aptamer sequence on the target RNA. Curr. Protoc. Cell Biol. 37:17.11.1‐17.11.20. © 2007 by John Wiley & Sons, Inc.

Keywords: protein complementation; aptamer‐protein interactions; RNA localization; fluorescent proteins; eukaryotic initiation factor 4A; bacterial cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Design and Cloning of DNA Contructs for the Expression of Protein and RNA Components of the Complementation Complex
  • Basic Protocol 2: Expression of RNA Labeling Components in E. Coli
  • Basic Protocol 3: Analysis of Cells by Flow Cytometry
  • Basic Protocol 4: Analysis of Cells by Microscopy
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Design and Cloning of DNA Contructs for the Expression of Protein and RNA Components of the Complementation Complex

  Materials
  • ThermoPol reaction buffer (New England Biolabs)
  • 10 mM dNTP solution (containing all four dNTPs; see appendix 3F, but prepare at 10 mM)
  • 10 mM primers (see Table 17.11.1)
  • EGFP template: DNA encoding EGFP (Clontech)
  • Vent exo DNA polymerase (New England Biolabs)
  • eIF4AI template: DNA encoding mouse eIF4AI (plasmid pGEX‐4AI; available from Chris Proud, University of Dundee, U.K., )
  • 1% agarose gel (Voytas, 2000)
  • PCR purification kit (QIAquick PCR Purification Kit, Qiagen)
  • Appropriate restriction enzymes (NcoI, BamHI, SalI, NotI, DpnI, XboI, XhoI; New England Biolabs)
  • DNA purification kit (QIAprep Spin Miniprep Kit, Qiagen)
  • Cloning vectors: pACYCDuet 1 or pETDuet‐1 (Novagen)
  • T4 DNA ligase and 10× buffer
  • Competent E. coli cells (also see Seidman et al., ): e.g., XL‐10 (Stratagene), DH5αPRO (Clontech)
  • LB plates and liquid medium ( appendix 2A) containing appropriate antibiotic
  • Thermostable DNA polymerase appropriate for amplifying long templates (e.g., DNA polymerase from Expand Long Template PCR System; Roche Diagnostics) and corresponding 10× buffer
  • Pfu Turbo DNA Polymerase (Stratagene) and corresponding 10× buffer
  • Thermal cycler
  • 16° and 65°C water baths
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, 2000), transformation of E. coli (Seidman et al., ), DNA miniprep from bacteria by alkaline lysis (Wilson, 2001), and gel purification of DNA (Moore et al., )
    Table 7.1.1   MaterialsPrimers for Amplifying Gene Fragments for Cloning

    Primers Sequence (5′ to 3′) a
    GFP‐A1 CCCGACCATGGTGAGCAAGGGCGAGGAGCTGTTC
    GFP‐A2 CCCGAGGATCCCTGCTTGTCGGCCATGATATAGAC
    GFP‐B1 CCCGACCATGGGCAAGAACGGCATCAAGGTGAAC
    GFP‐B2 CCGAGGATCCCTTGTACAGCTCGTCCATGCCGA
    eIF4A‐F1‐1 CCCGAGTCGACATGGAGCCGGAAGGCGTCATCGA
    eIF4A‐F1‐2 CGAGCGGCCGCTCAAGGGTCTCTCATAAATTTCTT
    eIF4A‐F2‐1 CCCGAGTCGACATTCGGATTCTTGTCAAGAAGGAAG
    eIF4A‐F2‐2 CGAGCGGCCGCTCAAATGAGGTCAGCAACGTTGAG

     aUnderlined bases correspond to restriction enzyme sites used for cloning into appropriate vectors.

Basic Protocol 2: Expression of RNA Labeling Components in E. Coli

  Materials
  • Competent E. coli cells (also see Seidman et al., ): e.g., BL21(DE)3 (Stratagene)
  • Plasmid encoding protein fusions ( protocol 1, step 29)
  • Plasmid encoding tagged RNA ( protocol 1, step 34)
  • LB plates ( appendix 2A) containing appropriate antibiotic
  • LB medium ( appendix 2A) containing appropriate antibiotic
  • LB medium ( appendix 2A) containing 1 mM IPTG
  • Additional reagents and equipment for transformation of E. coli (Seidman et al., )

Basic Protocol 3: Analysis of Cells by Flow Cytometry

  Materials
  • Cells expressing protein and RNA components of the complementation complex ( protocol 2, step 1)
  • Cells expressing only protein fusions (Control 1; protocol 2, step 29)
  • Cells where protein expression have not been turned on (lacking IPTG, Control 2; perform steps 1 to 4 of protocol 1, but omit IPTG in step 4)
  • Cells expressing protein fusions and an untagged RNA template (pET plasmid, Novagen)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Flow cytometer with 488‐nm excitation filter for collection of green fluorescence

Basic Protocol 4: Analysis of Cells by Microscopy

  Materials
  • Agarose
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Cells expressing protein and RNA component of the complementation complex ( protocol 2, steps 3 to 4)
  • Cells expressing only protein fusions (Control 1; protocol 1, step 29)
  • 50°C water bath
  • Vacuum aspirator
  • 15‐well multi‐test microscope slides (e.g., MP Biomedicals)
  • Long (e.g., 24 × 50–mm) coverslips
  • Inverted fluorescence microscope allowing excitation at 490 to 500 nm and emission detection at 525 to 535 nm, equipped with neutral density (ND) filters
  • Additional reagents and equipment for fluorescence microscopy (unit 4.2) and differential interference contrast microcopy (unit 4.1)
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Figures

Videos

Literature Cited

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