SELEX-Based In Vitro Binding Assay to Identify RNA-Protein Interactions

Abstract

Source: Singh, R. Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins. J. Vis. Exp. ( (2019).

In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.

Protocol

1. Protein binding reaction and separation of bound RNA

1. Carry out binding of protein and RNA in 10 mM Tris-HCl, pH 7.5 in a volume of 100 µL by adding the following ingredients to these final concentrations: 50 mM KCl, 1 mM DTT, 0.09 µg/µL bovine serum albumin, 0.5 units/µL RNasin, 0.15 µg/µL tRNA, 1 mM EDTA, and 30 µL of appropriate recombinant protein (PTB) concentration. Add RNA from the appropriate pool.
   NOTE: The splicing factor U2AF65 typically binds to the polypyrimidine-tract/3' splice sites of model introns with a binding affinity (equilibrium dissociation constant or ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gel Electrophoresis equipment	Standard	Standard
Nitrocellulose	Millipore	HAWP
Nitrocellulose	Schleicher & Schuell	PROTRAN
Polyacrylamide gel solutions	Standard	Standard
Proteinase K	NEB	P8107S
Recombinant PTB	Laboratory Preparation	Not applicable
Reverse Transcriptase	NEB	M0277S
Vacuum manifold	Fisher Scientific	XX1002500	Millipore 25mm Glass Microanalysis Vacuum Filter
Vacuum manifold	Millipore	XX2702552	1225 Sampling Vacuum Manifold
X-ray films	Standard	Standard
Glass Plates	Standard	Standard