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

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

Fareh, M. (TU Delft BN/Chirlmin Joo Lab; Kavli institute of nanoscience Delft)
Loeff, L. (TU Delft BN/Chirlmin Joo Lab; Kavli institute of nanoscience Delft)
Szczepaniak, M. (TU Delft BN/Chirlmin Joo Lab; Kavli institute of nanoscience Delft)
van Eijkeren-Haagsma, A.C. (TU Delft BN/Technici en Analisten; Kavli institute of nanoscience Delft)
Yeom, K.H. (TU Delft BN/Chirlmin Joo Lab; Kavli institute of nanoscience Delft)
Joo, C. (TU Delft BN/Chirlmin Joo Lab; Kavli institute of nanoscience Delft)

2016

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.

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

http://resolver.tudelft.nl/uuid:dd841cf9-aeac-4169-b8d3-8d7683b38548

https://doi.org/10.1016/j.ymeth.2016.03.022

2017-03-25

1046-2023

Methods, 105, 99-108

Accepted Author Manuscript

Institutional Repository

journal article

© 2016 M. Fareh, L. Loeff, M. Szczepaniak, A.C. van Eijkeren-Haagsma, K.H. Yeom, C. Joo