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

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

doi:10.1016/j.ymeth.2016.03.022

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

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

Department of Physics

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

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 language	English
Pages (from-to)	99-108
Number of pages	10
Journal	Methods
Volume	105
DOIs	https://doi.org/10.1016/j.ymeth.2016.03.022
State	Published - 1 Aug 2016

Bibliographical note

Funding Information:
C.J. was funded by European Research Council under the European Union’s Seventh Framework Programme [FP7/2007-2013]/ERC grant agreement n° [ 309509 ]. We thank V. Narry Kim (Seoul National University), Mikiko Siomi (Keio University), and John Strouboulis (B.S.R.C. Alexander Fleming) for sharing plasmids. We thank Joo lab members for their help.

Funding Information:
C.J. was funded by European Research Council under the European Union's Seventh Framework Programme [FP7/2007-2013]/ERC grant agreement n? [309509]. We thank V. Narry Kim (Seoul National University), Mikiko Siomi (Keio University), and John Strouboulis (B.S.R.C. Alexander Fleming) for sharing plasmids. We thank Joo lab members for their help.

Publisher Copyright:
© 2016 Elsevier Inc.

Keywords

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

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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.",

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note = "Funding Information: C.J. was funded by European Research Council under the European Union{\textquoteright}s Seventh Framework Programme [FP7/2007-2013]/ERC grant agreement n° [ 309509 ]. We thank V. Narry Kim (Seoul National University), Mikiko Siomi (Keio University), and John Strouboulis (B.S.R.C. Alexander Fleming) for sharing plasmids. We thank Joo lab members for their help. Funding Information: C.J. was funded by European Research Council under the European Union's Seventh Framework Programme [FP7/2007-2013]/ERC grant agreement n? [309509]. We thank V. Narry Kim (Seoul National University), Mikiko Siomi (Keio University), and John Strouboulis (B.S.R.C. Alexander Fleming) for sharing plasmids. We thank Joo lab members for their help. Publisher Copyright: {\textcopyright} 2016 Elsevier Inc.",

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

Abstract

Bibliographical note

Keywords

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