Single-molecule pull-down for investigating protein–nucleic acid interactions

Mohamed Fareh, Luuk Loeff, Malwina Szczepaniak, Anna C. Haagsma, Kyu Hyeon Yeom, Chirlmin Joo

Research output: Contribution to journalArticleScientificpeer-review

11 Citations (Scopus)
61 Downloads (Pure)

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.

Original languageEnglish
Pages (from-to)99-108
Number of pages10
JournalMethods
Volume105
DOIs
Publication statusPublished - 2016

Keywords

  • Dicer
  • Drosha
  • Protein complex
  • RNA interference
  • Single-molecule fluorescence
  • Single-protein pull-down
  • TUT4

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