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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
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Probing RNA-Protein Interactions with Single-Molecule Pull-Down Assays.

Mohamed Fareh1,2, Chirlmin Joo3

  • 1Department of BioNanoScience, Kavli Institute of NanoScience, Delft University of Technology, Building 58, vander Maasweg 9, Delft, 2629 HZ, The Netherlands. m.fareh@tudelft.nl.

Methods in Molecular Biology (Clifton, N.J.)
|June 30, 2018
PubMed
Summary

New single-molecule techniques enable real-time observation of RNA-protein interactions. This study details protocols for studying ribonucleoprotein complexes, like microRNA biogenesis, with high resolution.

Keywords:
Protein complexRNA labelingRNA ligationSingle protein pull-downSingle-molecule fluorescence

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysics

Background:

  • Single-molecule techniques offer high spatiotemporal resolution for observing molecular interactions.
  • Studying eukaryotic protein complexes and labeled RNAs at the single-molecule level presents challenges.

Purpose of the Study:

  • To develop and present protocols for studying RNA-interacting protein complexes using single-molecule fluorescence and pull-down assays.
  • To provide a framework for investigating the function of ribonucleoprotein complexes in real time.

Main Methods:

  • Combining single-molecule fluorescence with protein complex pull-down techniques.
  • Detailed protocols for RNA labeling, protein complex purification, and single-molecule imaging.
  • Development of novel single-molecule assays with sub-second time resolution.

Main Results:

  • Demonstrated utility of the single-molecule approach using human Dicer and TRBP in microRNA biogenesis.
  • Successfully visualized the real-time orchestration of microRNA biogenesis.
  • Established protocols applicable to various essential ribonucleoprotein complexes.

Conclusions:

  • The developed single-molecule pull-down and fluorescence assays are effective for studying RNA-protein interactions.
  • These methods provide crucial insights into the dynamics of ribonucleoprotein complexes.
  • The protocols are versatile and can be applied to a wide range of cellular processes.