Single-molecule pull-down for investigating protein–nucleic acid interactions
Mohamed Fareh (TU Delft - BN/Chirlmin Joo Lab, Kavli institute of nanoscience Delft)
Luuk Loeff (Kavli institute of nanoscience Delft, TU Delft - BN/Chirlmin Joo Lab)
Malwina Szczepaniak (TU Delft - BN/Chirlmin Joo Lab, Kavli institute of nanoscience Delft)
Anna C. Haagsma (Kavli institute of nanoscience Delft, TU Delft - BN/Technici en Analisten)
Kyu Hyeon Yeom (TU Delft - BN/Chirlmin Joo Lab, Kavli institute of nanoscience Delft)
Chirlmin Joo (TU Delft - BN/Chirlmin Joo Lab, Kavli institute of nanoscience Delft)
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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.